Methods and products related to evaluating the quality of a polysaccharide

ABSTRACT

The invention relates to methods and products for characterizing and using polysaccharides. Low molecular weight heparin products and methods of use are described. Methods for characterizing purity and activity of polysaccharide preparations including glycosaminoglycans such as heparin are also described.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/183323, filed Jul. 15, 2005, which is a divisional of U.S. patentapplication Ser. No. 09/951138, filed Sep. 12, 2001, which claimspriority under 35 U.S.C. §119 from U.S. provisional application Ser. No.60/231994, filed Sep. 12, 2000, the entire contents of each of which isincorporated herein by reference.

GOVERNMENT SUPPORT

The present invention was supported in part by a grant from the UnitedStates National Institutes of Health under contract/grant number GM57073. The U.S. Government may retain certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to methods and products associated withcharacterizing and using polysaccharides. In particular low molecularweight heparin products and methods of using these products aredescribed. Methods for characterizing purity and activity ofpolysaccharide preparations including glycosaminoglycans such as heparinare also described.

BACKGROUND OF THE INVENTION

Coagulation is a physiological pathway involved in maintaining normalblood hemostasis in mammals. Under conditions in which a vascular injuryoccurs, the coagulation pathway is stimulated to form a blood clot toprevent the loss of blood. Immediately after the vascular injury occurs,blood platelets begin to aggregate at the site of injury forming aphysical plug to stop the leakage. In addition, the injured vesselundergoes vasoconstriction to reduce the blood flow to the area andfibrin begins to aggregate forming an insoluble network or clot, whichcovers the ruptured area. When an imbalance in the coagulation pathwayshifts towards excessive coagulation, the result is the development ofthrombotic tendencies, which are often manifested as heart attacks,strokes, deep vein thrombosis, and myocardial infarcts. Currenttherapies for treating disorders associated with imbalances in thecoagulation pathway involve many risks and must be carefully controlled.

Heparin, a highly sulphated heparin-like glycosaminoglycan (HLGAG)produced by mast cells, is a widely used clinical anticoagulant, and isone of the first biopolymeric drugs and one of the few carbohydratedrugs. Heparin primarily elicits its effect through two mechanisms, bothof which involve binding of antithrombin III (AT-III) to a specificpentasaccharide sequence, H_(NAc/S,6S)GH_(NS,3S,6S)I_(2S)H_(NS,6S)contained within the polymer. First, AT-III binding to thepentasaccharide induces a conformational change in the protein thatmediates its inhibition of factor Xa. Second, thrombin (factor IIa) alsobinds to heparin at a site proximate to the pentasaccharide AT-IIIbinding site. Formation of a ternary complex between AT-III, thrombinand heparin results in inactivation of thrombin. Unlike its anti-Xaactivity that requires only the AT-III pentasaccharide-binding site,heparin's anti-IIa activity is size-dependant, requiring at least 18saccharide units for the efficient formation of an AT-III, thrombin, andheparin ternary complex.

In addition to heparin's anticoagulant properties, its complexity andwide distribution in mammals have lead to the suggestion that it mayalso be involved in a wide range of additional biological activities.Heparin-like glycosaminoglycans (HLGAGs), present both at the cellsurface and in the extracellular matrix, are a group of complexpolysaccharides that are variable in length, consisting of adisaccharide repeat unit composed of glucosamine and an uronic acid(either iduronic or glucuronic acid). The high degree of complexity forHLGAGs arises not only from their polydispersity and the possibility oftwo different uronic acid components, but also from differentialmodification at four positions of the disaccharide unit. Threepositions, viz., C2 of the uronic acid and the C3, C6 positions of theglucosamine can be O-sulfated. In addition, C2 of the glucosamine can beN-acetylated or N-sulfated. Together, these modifications couldtheoretically lead to 32 possible disaccharide units, making HLGAGspotentially more information dense than either DNA (4 bases) or proteins(20 amino acids). This enormity of possible structural variants allowsHLGAGs to be involved in a large number of diverse biological processes,including angiogenesis (Sasisekharan, R., Moses, M. A., Nugent, M. A.,Cooney, C. L. & Langer, R. (1994) Proc Natl Acad Sci USA 91, 1524-8.),embryogenesis (Binari, R. C., Staveley, B. E., Johnon, W. A., Godavarti,R., Sasisekharan, R. & Manoukian, A. S. (1997) Development 124, 2623-32;Tsuda, M., Kamimura, K., Nakato, H., Archer, M., Staatz, W., Fox, B.,Humphrey, M., Olson, S., Futch, T., Kaluza, V., Siegfried, E., Stam, L.& Selleck, S. B. (1999) Nature 400, 276-80.; and Lin, X., Buff, E. M.,Perrimon, N. & Michelson, A. M. (1999) Development 126, 3715-23.) andthe formation of β-fibrils in Alzheimer's disease (McLaurin, J.,Franklin, T., Zhang, X., Deng, J. & Fraser, P. E. (1999) Eur J Biochem266, 1101-10. And Lindahl, B., Westling, C., Gimenez-Gallego, G.,Lindahl, U. & Salmivirta, M. (1999) J Biol Chem 274, 30631-5).

Although heparin is highly efficacious in a variety of clinicalsituations and has the potential to be used in many others, the sideeffects associated with heparin therapy are many and varied. Sideeffects such as heparin-induced thrombocytopenia (HIT) are primarilyassociated with the long chain of un-fractionated heparin (UFH), whichprovides binding domains for various proteins. This has lead to theexplosion in the generation and utilisation of low molecular weightheparin (LMWH) as an efficacious alternative to UFH. Although attentionhas been focused on LMWH as heparin substitutes due to their morepredictable pharmacological action, reduced side effects, sustainedantithrombotic activity, and better bioavailability, there is at presentlimited ability to standardize the LMWH manufacturing process. Becausethe LMWH are derived from heparins and hence are polydisperse andmicroheterogenous, with undefined structure, they possess inherentvariability, which currently prevents an efficient process for theirmanufacture. It would be of value both medically and scientifically tohave a consistent, quality controlled, time efficient, concentrationindependent, and highly reproducible method for producing heparin andother glycosaminoglycans.

In an attempt to characterize the molecular, structural, and activityvariations of heparin, several techniques have been investigated for theanalysis of heparin preparations. Gradient polyacrylamide gelelectrophoresis (PAGE) and strong ion exchange HPLC (SAX) have been usedfor the qualitative and quantitative analysis of heparin preparations.Although the gradient PAGE method can be useful in determining molecularweight, it suffers from the lack of resolution, particularly the lack ofresolution of different oligosaccharides having identical size.SAX-HPLC, which relies on detection by ultraviolet absorbance, is ofteninsufficiently sensitive for detecting small amounts of structurallyimportant heparin-derived oligosaccharides. The current technologies forpurifying and analyzing heparins and other glycosaminoglycans areinsufficient. There is a great clinical and scientific need for improvedisolation and analysis methods.

SUMMARY OF THE INVENTION

The invention relates in some aspects to methods for characterizingpolysaccharide preparations. As a result of the complex saccharidestructures, it has been difficult if not impossible to characterize thepurity and/or activity of polysaccharide preparations. Unlike nucleicacid and protein samples, polysaccharide preparations are generallycharacterized based on their ability to produce a certain level ofactivity in a biological sample. These assays do not achieve the levelof accuracy that can be achieved by direct structural characterization.According to some aspects of the invention a method of analyzing andcharacterizing a polysaccharide sample is provided. The method involvesapplying an experimental constraint to a polysaccharide in a sample toproduce a modified polysaccharide having a signature component,detecting the presence of the signature component in the sample as anindication that the polysaccharide is present in the sample, anddetermining the presence or absence of the signature component toanalyze the sample. In some embodiments the signature component has aknown biological activity and in other embodiments the signaturecomponent is biologically inactive.

The experimental constraint applied to the sample is any type ofmanipulation that results in the identification of the presence orabsence of the signature component. The experimental constraint may, forexample, be any one alone or combination of the following types ofexperimental constraints: capillary electrophoresis, high pressureliquid chromatography, gel permeation chromatography, nuclear magneticresonance, modification with an enzyme such as digestion with anexoenzyme or an endoenzyme, chemical digestion, or chemicalmodification.

The signature component can be used to provide information about thesample. Some of the uses depend on whether the signature component is anactive or inactive biological component. For instance, in some caseswhen the signature component is an active biological component and thesample is a batch of polysaccharide, the signature component may be usedto monitor the purity of the batch by determining the amount ofsignature component in the batch. In other embodiments the method ofanalysis is a method for monitoring the presence of active components inthe sample, wherein the presence of the signature component in thesample is indicative of an active component in the sample. In otherembodiments the method of analysis is a method for determining theamount of active components in the sample by determining the amount ofsignature component in the sample. The method may also be performed onat least two samples such that the relative amounts of signaturecomponent in each of the at least two samples is determined, and thehighest relative level of signature component is indicative of the mostactive sample.

In some instances when the signature component is an inactive biologicalcomponent, the method of analysis may be a method for monitoring thepresence of active components in the sample, wherein the presence of thesignature component in the sample is indicative of a sample lacking anactive component.

The methods are also useful in some embodiments for identifyingbiologically active molecules. For instance, the signature component maybe used to screen a library.

Thus in some embodiments the signature component is a biologicallyactive portion of a polysaccharide. Biologically active portions ofpolysaccharides include but are not limited to a tetrasaccharide of theAT-III biding domain of heparin, a tetrasaccharide of the FGF bidingdomain of heparin, ΔUH_(NAC,6S)GH_(NS,3S,6S); ΔUH_(NS,6S)GH_(NS,3S,6S);ΔUH_(NAC,6S)GH_(NS,3S); and ΔUH_(NS,6S)GH_(NS,3S).

The polysaccharide in some embodiments is a glycosaminoglycan, such as alow molecular weight heparin (LMWH), heparin, a biotechnologicallyprepared heparin, a chemically modified heparin, a synthetic heparin,and a heparan sulfate.

In another embodiment the polysaccharide in the sample is compared to areference database of polysaccharides of identical size as thepolysaccharide, wherein the polysaccharides of the reference databasehave also been subjected to the same experimental constraints as thepolysaccharide in the sample, wherein the comparison provides acompositional analysis of the sample polysaccharide.

In some preferred embodiments the sample is a pharmaceutical product. Inother embodiments the sample is biological sample, such as a bloodsample.

A method for evaluating the quality of a polysaccharide sample isprovided according to other aspects of the invention. The methodinvolves identifying a component within the polysaccharide sample,determining a quantitative value of the amount of component, wherein thequantitative value of the component is indicative of the quality of thepolysaccharide sample. In one embodiment the method involves identifyingat least two components within the polysaccharide sample and determininga quantitative value of the amount of each of the at least twocomponents to evaluate the quality of the polysaccharide sample.

The quantitative value may be calculated by a variety of differentmethods, depending on how the sample is processed to identify thecomponent. For instance, the quantitative value may be calculated as thearea under the curve when the sample is processed by capillaryelectrophoresis, as the response factor, or as the percent relativeamount of each fraction present in the sample.

In one embodiment the step of calculating the percent relative amount ofeach fraction present in the sample is determined according to the belowequation:PRA=RF×AUC_(%R)wherein

-   -   PRA=percent relative amount of each fraction    -   RF=response factor    -   AUC_(%R)=percent relative AUC [(100×AUC_(C))/AUC_(T))]    -   AUC_(C)=Area under the curve for one component    -   AUC_(T)=the sum of the Area under the curve for all components.

In another embodiment computer-implemented method for generating a datastructure, tangibly embodied in a computer-readable medium, representinga quantitative value of a component of a polysaccharide, the methodcomprising an act of performing the above calculation.

In one embodiment the component is signature componentΔUH_(NAC,6S)GH_(NS,3S,6S), ΔUH_(NS,6S)GH_(NS,3S,6S);ΔUH_(NAC,6S)GH_(NS,3S); or ΔUH_(NS,6S)GH_(NS,3S).

In another aspect the invention relates to a method of producing acomposition of glycosaminoglycans. The method involves performing a saltprecipitation of a glycosaminoglycan containing sample in a solvent toproduce a first higher molecular weight fraction, and a second fractionof isolated LMWH, and processing the second fraction of isolated LMWH toproduce a concentrated LMWH preparation. In a preferred embodiment thesalt used in the precipitation step is a salt of divalent cations andweak anions. The prior art generally taught that when a method forisolating heparin using a salt precipitation is used, the first fractionshould be processed to generate LMWH and the second fraction should bediscarded. It has been discovered that the second fraction actuallycontains a preferred source of biologically active LMWH.

In some embodiments the salt of the divalent cations and weak anions isselected from among the group comprising; barium, calcium, magnesium,strontium, copper, nickel, cadmium, zinc., mercury, beryllium, nickel,palladium, platinum, iron, and tin. In other embodiments the salt ofdivalent cations and weak anions are acetates of cations of elements ofthe periodic table having divalent valence.

The components of the LMWH fraction can be further altered bymanipulating the temperature and type of solvent used in theprecipitation. For instance, in one embodiment the precipitation may beperformed at a temperature in a range of 0° C. to 70° C. In otherembodiments the temperature of the mixture is 70° C., 60° C., 50° C.,40° C., 30° C. 25° C., 20° C., 15° C., 10° C., 5° C., 3° C., 2° C., 1°C., or 0° C. In yet other embodiments the precipitation is performed ata temperature of 4° C. Preferably the solvent is a polar solvent. Polarsolvents include but are not limited to H₂O, H₂O mixed with ethanol, H₂Omixed with acetone, or a combination of H₂O, ethanol, and acetone. Insome embodiments the polar solvent used in the precipitation has avolume to volume H₂O: ethanol ratio in the range of 99:1, 95:5, 90:10,85:15, 80:20, 75:25, 65:35, 55:45, 50:50, 45:55, 35:65, 25:75, 20:80,15:85, 10:90, 5:95, or 1:99. In other embodiments the polar solvent usedin the precipitation has a volume to volume H₂O: acetone ratio in therange of 99:1, 95:5, 90:10, 85:15, 80:20, 75:25, 65:35, 55:45, 50:50,45:55, 35:65, 25:75, 20:80, 15:85, 10:90, 5:95, or 1:99. In oneembodiment the polar solvent is a mixture of H₂O, ethanol, and acetone.

The step of processing of the second fraction to yield the concentratedLMWH preparation, in one embodiment, is an enzymatic digestion, such asdigestion with Heparinase III. In other embodiments, the processing ofthe second fraction to yield the concentrated LMWH preparation ischemical degradation. In some embodiments the method of chemicaldegradation is selected from the group including oxidativedepolymerization with H₂O₂ or CU⁺ and H₂O₂, deaminative cleavage withisoamyl nitrite, or nitrous acid, β-eliminative cleavage with benzylester of heparin by alkaline treatment or by heparinase. The processingof the second fraction to yield the concentrated LMWH preparation inother embodiments is a purification step to produce a purified LMWHpreparation. The method may involve the further step of formulating thepurified LMWH preparation in a pharmaceutical carrier.

In other embodiments the glycosaminoglycan is selected from the groupconsisting of heparin, heparin analogs, LMWH, biotechnological heparin,chemically modified heparin, or synthetic heparin.

Compositions comprising a LMWH preparation having an anti-Xa activity ofat least 150 IU/mg and/or LMWH preparation having an anti-factorXa:anti-factor IIa activity ratio of greater than 5 are providedaccording to other aspects of the invention. In some embodiments theLMWH preparation is isolated and in other embodiments it is synthetic.In some embodiments the LMWH preparation having an anti-Xa activity ofat least 150 IU/mg has an anti-factor Xa:anti-factor Ia activity ratioof at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In other aspects the composition is a LMWH preparation having at least15% disulfated disaccharides, less than 75% trisulfated disaccharides,3-5% monosulfated disaccharides, and at least 2% 4-7 tetrasaccharide.

The composition may be formulated for therapeutic delivery to a subjectby methods such as subcutaneous delivery, intravenous delivery, aerosoldelivery, or oral delivery.

In some embodiments the LMWH preparation includes at least 3.5%, 4.0%,or 5.0% ΔUH_(NAC,6S)GH_(NS,3S,6S), ΔUH_(NS,6S)GH_(NS,3S,6S);ΔUH_(NAC,6S)GH_(NS,3S); or ΔUH_(NS,6S)GH_(NS,3S).

A method for treating a subject having a condition is provided accordingto other aspects of the invention. The method involves selecting acomposition of LMWH having an identified level of AT-binding sequence,the level of AT-binding sequence selected depending on the condition tobe treated in the subject, and administering to a subject an effectiveamount of the composition of LMWH having an identified level ofAT-binding sequence.

The subject, in some embodiments, has or is at risk of developing venousor arterial thromboembolic disease. The LMWH preparation administered tothese subjects may include at least 3.5%, 4.0%, or 5.0%ΔUH_(NAC,6S)GH_(NS,3S,6S), ΔUH_(NS,6S)GH_(NS,3S,6S);ΔUH_(NAC,6S)GH_(NS,3S); or ΔUH_(NS,6S)GH_(NS,3S). In other embodimentsthe composition of LMWH is a LMWH preparation having an anti-Xa activityof at least 150 IU/mg. In yet other embodiments the composition of LMWHis a LMWH preparation having an anti-factor Xa:anti-factor IIa activityratio of greater than 5.

The invention in other aspects includes a composition, comprising, aLMWH preparation prepared by a process comprising: obtaining a heparinpreparation, and performing an exhaustive digestion of the heparinpreparation using heparinase I, heparinase II, and heparinase III.

In other aspects the invention relates to a kit for analyzing apolysaccharide sample including a control composition for identifying asignature component of a polysaccharide, and instructions for applyingan experimental constraint to a polysaccharide sample to produce amodified polysaccharide having a signature component characteristic ofthe polysaccharide and for comparing the modified polysaccharide to thecontrol composition to identify the presence or absence of the signaturecomponent. In some embodiments the kit also includes a composition forapplying an experimental constraint to the polysaccharide sample, suchas, for example, an exoenzyme or an endoenzyme. In yet other embodimentsthe instructions include the steps for quantifying the signaturecomponent of the polysaccharide in the sample.

Each of the limitations of the invention can encompass variousembodiments of the invention. It is, therefore, anticipated that each ofthe limitations of the invention involving any one element orcombinations of elements can be included in each aspect of theinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a MALDI mass spectrum of the protonated complex of AT-10 with(RG)₁₉R.

FIG. 2 depicts Heparinase treatment of AT-10. (A) Incomplete heparinaseI treatment of AT-10. Under the conditions used in this study,heparinase I cleaves a glycosidic linkage containing an I_(2S). (B)MALDI mass spectrum of AT-10 fragments from exhaustive digestion withheparinase I. (C) MALDI mass spectrum of tagged AT-10 treated withheparinase I shows five fragments: one with molecular mass of 576.7 Da(assignable to ±D), two tetrasaccharides with molecular mass of 1037.9(*) and 1154.0 Da, and a mass tagged hexasaccharide with a molecularmass of 1671.4 (mass of 1615.3 plus the mass tag of 56.1). Since thecoupling efficiency was ˜90%, also seen is unlabeled hexasaccharide(mass of 1615.1).

FIG. 3 depicts exhaustive degradation of AT-10 with nitrous acid.Nitrous acid cleaves at H_(NS) residues, leaving behind ananhydromannose (Δ=97.1 Da). Shown in the inset is the mass spectrum ofthe degradation profile when the sample was treated with iduronidase(top), glucosamine 6-O sulfatase (middle) and glucuronidase (bottom) inthat order.

FIG. 4 is a MALDI mass spectrum showing partial nitrous acid degradationof AT-10.

FIG. 5 shows the structures of the three oligosaccharide model compoundsused in this study. (A) Pentasaccharide 1 (Penta 1) has the sequenceH_(NS,6S)GH_(NS,3S,6S)I_(2S)H_(NS,6S,OMe), contains a fully intactAT-III binding site and has a calculated molecular mass of 1508.2. Thetwo glycosidic linkages potentially susceptible to heparinase I, II, orIII cleavage are labeled A.1 and A.2. (B) Pentasaccharide 2 (Penta 2),with the sequence H_(NS,6S)GH_(NS,6S)I_(2S)H_(NS,6S,OMe) and acalculated molecular mass of 1428.1, is structurally identical to Penta1, less a 3-O sulfate on the internal glucosamine, thus it does notcontain a full AT-III site. As with Penta 1 the bonds potentiallysusceptible to heparinase cleavage are marked B.1 and B.2. (C) Aheparinase-derived hexasaccharide (Hexa 1), with the sequenceΔU_(2S)H_(NS,6S)IH_(NAc,6S),GH_(NS,3S,6S), was also used in this study.Hexa 1 (calculated molecular mass 1614.3) contains only a partiallyintact AT-III binding site; similar to AT-10 it is missing the reducingend I_(2S)H_(NS,6S) disaccharide unit. As with Penta 1 and Penta 2,sites of potential cleavage are marked C.1, C.2.

FIG. 6 is a MALDI mass spectra of (A) heparinase I, (B) heparinase II,and (C) heparinase III digestion products of Penta 1. Both heparinase Iand II clip Penta 1 at the G_(NS,3S,6S)⇓I_(2S)H_(NS,6S) linkage (siteA.2) to yield a pentasulfated trisaccharide and a trisulfateddisaccharide product. Penta 1 is not cleavable by heparinase III.

FIG. 7 is a MALDI mass spectra of (A) heparinase I, (B) heparinase II,and (C) heparinase III digestion products of Penta 2 complexed with(RG)₁₉R.

FIG. 8 is a MALDI mass spectra of (A) heparinase I, (B) heparinase II,and (C) heparinase III digestion products of Hexa 1 complexed with(RG)₁₉R.

FIG. 9 shows fluorescence titration of AT-III with either full lengthheparin (●) or AT-10 (♦) at pH 6.0 I=0.025. Data is plotted as the ratioof AT-III fluorescence upon the introduction of saccharide to theinitial AT-III fluorescence (I/I_(o)) vs. concentration of addedsaccharide. The data was fitted by nonlinear regression and the K_(D)determined. For heparin the measured K_(D) value was 10 nM, whereas forAT-10 this value was 800 nM. The inset shows the binding of heparin toAT-III at pH 7.4 I=0.15. The measured K_(D) of 36 nM agrees favorablywith other determinations of the affinity of heparin for AT-III.

FIG. 10 shows Functional analysis of the AT-10 decasaccharide andcomparison to the AT-III binding pentasaccharide. The in vitroanticoagulant activity of the AT-10 decasaccharide (σ) was compared toboth the synthetic pentasaccharide (▪) or enoxaparin (◯), a lowmolecular weight heparin generated through chemical cleavage of heparin.The activities of the three compounds was assessed by measuring either(A) anti-IIa activity, (B) anti-Xa activity, (C) anti-Xa activity usingpurified factor Xa or (D) via HepTest. Also the activated partialthromboplastin time (APTT) and the prothrombin time (PT) was measuredwherein none of the compounds displayed significant activity, consistentwith their high ratio of anti-Xa:anti-IIa activity.

FIGS. 11A and 11B show graphs of compositional analysis of UFH derivedfrom porcine intestinal mucosa. UFH was digested with Heparinase I, II,and III and subjected to Capillary Electrophoresis (CE). Peak 1 (FIG.9A) was thus confirmed as the trisulfated disaccharideΔU_(2S),H_(NS,6S). Peaks 2, 3, and 4 are disulfated disaccharides, and5, 6, and 7 are monosulfated disaccharides. Peak 8 is thetetrasaccharide ΔUH_(NAC,6S)GH_(NS,3S,6S). In addition to these, thereis a small amount of unsulfated disaccharides migrating much slower thanthe sulfated saccharides, as shown in FIG. 9B.

FIG. 12 shows the CE trace of the exhaustive digest of AT-10pentasaccharide ΔU_(2S)H_(NS,6S) ΔU_(2S)H_(NS,6S)ΔU_(2S)H_(NS,6S)IH_(NAC,6S) GH_(NS,3S,6S). The tetrasaccharide of peak 8in the exhaustive digest of heparin has the same mass, and migrationtime as ΔUH_(NAC,6S)GH_(NS,3S,6S).

FIG. 13 is a graph of anti-factor Xa activity for different fractions ofUFH as a function of their ΔUH_(NAC,6S)GH_(NS,3S,6S) content. A plot ofanti-factor Xa activity as a function of % ΔUH_(NAC,6S)GH_(NS,3S,6S)gives a straight line with r=0.91.

DETAILED DESCRIPTION

The invention involves several discoveries that have led to new advancesin the field of polysaccharide biology. One of the major problems incharacterizing polysaccharides results from their structural diversity.This structural diversity is one of the factors that has made itdifficult to study sequence-function relationships for polysaccharides.Chemical synthesis of defined oligosaccharides has been used in studyingthe relative contribution to biological activities, such as the highaffinity AT-III binding of specific modifications in the pentasaccharidesequence of heparin (Desai, U. R., Petitou, M., Bjork, I. & Olson, S. T.(1998)J Biol Chem 273, 7478-87.). However, such synthetic methods arecomplex and have not been widely applied to the study of otherbiological sequences. An alternative approach involving affinityfractionation of polysaccharide with proteins of interest and subsequentcharacterisation has provided some overall information regardingsulfation patterns of polysaccharides that determine affinity(Parthasarathy, N., Gotow, L. F., Bottoms, J. D., Kute, T. E., Wagner,W. D). & Mulloy, B. (1998) J Biol Chem 273, 21111-4.; Sasaki, T.,Larsson, H., Kreuger, J., Salmivirta, M., Claesson-Welsh, L., Lindahl,U., Hohenester, E. & Timpl, R. (1999) Embo J 18, 6240-8.; and Kreuger,J., Prydz, K., Pettersson, R. F., Lindahl, U. & Salmivirta, M. (1999)Glycobiology 9, 723-9.).

The invention is based, in one aspect, on a new method forcharacterising samples of polysaccharides. It has been discovered thatpolysaccharide sequences can be rapidly and accurately sequenced toidentify a signature component of the polysaccharide. The signaturecomponent can be used to characterize the polysaccharide sample in waysthat were not previously possible. The analysis of pharmaceutical-gradepolysaccharides is governed by the United States Pharmacopia (USP) andother national pharmacopia. Generally, the types of analysis requiredfor polysaccharides are functional assays and in some cases very generalstructural assays. The assays that are currently being used to determinethe activity/purity of a commercially available heparin preparation arean in vitro coagulation assay and a test for bacterial endotoxins. Theamount of heparin is determined to be that amount that will cause 1 mlof sheep plasma to half-clot when kept for 1 hour at 20° C. compared toa USP reference standard (defined as units/ml) or the FifthInternational standard for Unfractionated Heparin (WHO-5) (defined asInternational Units/ml). (Linhardt, R. J. & Gunay, N. S. (1999) SeminThromb Hemost 25, 5-16.). Compared with the strict regulatoryrequirements for other (non-polysaccharide) drugs these characterizationstandards are out of date.

The methods of the invention provide a much more accurate way forcharacterizing these samples. The methods involve manipulating apolysaccharide containing sample to identify the presence or absence ofa signature component. The amount of signature component present in thesample can be determined. The quantity of signature component gives anaccurate characterization of the sample.

Thus, in some aspects, the invention is a method of analyzing a sampleby applying an experimental constraint to a polysaccharide in a sample,to produce a modified polysaccharide, having a signature component,detecting the presence of the signature component, in the sample as anindication that the polysaccharide is present in the sample, anddetermining the presence or absence of the signature component toanalyze the sample.

A “polysaccharide” is a polymer composed of monosaccharides linked toone another. In many polysaccharides the basic building block of thepolysaccharide is actually a disaccharide unit, which can be repeatingor non-repeating. Thus, a unit when used with respect to apolysaccharide refers to a basic building block of a polysaccharide andcan include a monomeric building block (monosaccharide) or a dimericbuilding block (disaccharide).

The methods for characterizing polysaccharide samples were developedbased on experimental analysis of heparin-like glycosaminoglycans(HLGAGs) but the properties taught herein can be extended to otherpolysaccharides. The methods of the invention will be discussed withrespect to HLGAGs as an example, but the methods are not limited toHLGAGs. Thus in one embodiment the polysaccharide sample to be analysedincludes HLGAGs or glycosaminoglycans. As used herein the terms “HLGAG”and “glycosaminoglycans” are used interchangeably to refer to a familyof molecules having heparin like structures and properties. Thesemolecules include but are not limited to low molecular weight heparin(LMWH), heparin, biotechnologically prepared heparin, chemicallymodified heparin, synthetic heparin, and heparan sulfate. The term“biotechnological heparin” encompasses heparin that is prepared fromnatural sources of polysaccharides which have been chemically modifiedand is described in Razi et al., Bioche. J. 1995 Jul. 15; 309 (Pt 2):465-72. Chemically modified heparin is described in Yates et al.,Carbohydrate Res 1996 Nov. 20; 294:15-27, and is known to those of skillin the art. Synthetic heparin is well known to those of skill in the artand is described in Petitou, M. et al., Bioorg Med Chem Lett. 1999 Apr.19; 9(8):1161-6.

As shown in the Examples below the sequence of an AT-III fractionateddecasaccharide (AT-10), which may be used as a signature component ofHLGAGs, has been identified using a property-encoded nomenclature/massspectrometry scheme (PEN-MALDI), a sequencing methodology described inU.S. patent application Ser. Nos. 09/557,997 and 09/558,137 filed onApr. 24, 2000, having common inventorship, and Venkataraman, G.,Shriver, Z., Raman, R. & Sasisekharan, R. (1999) Science 286, 537-42.Integral Glycan Sequencing (IGS) (Tumbull, J. E., Hopwood, J. J. &Gallagher, J. T. (1999) Proc Natl Acad Sci USA 96, 2698-703.) and protonnuclear magnetic resonance (¹H NMR) analysis of the decasaccharide areconsistent with the results of PEN-MALDI. The flexibility of thissequencing strategy is also demonstrated by the fact that we can derivesequence information for contaminating oligosaccharides, if present.Sequencing of a chemically complex AT-III fractionated saccharide(including the rare 3-O-sulfation of glucosamine) established amethodology that can be extended to the analysis of other HLGAGoligosaccharides of interest, for example those HLGAGs with growthfactor binding properties. A straight-forward sequencing methodology forthese types of sequences has enabled structure-function studies of thisimportant class of molecules.

HLGAGs and other polysaccharides all have signature components. A“signature component” is an oligosaccharide which is present in andcharacteristic of a particular polysaccharide. The properties of thesignature selected for may depend on the type of polysaccharide beingstudied and the type of experimental constraint applied to thepolysaccharide. The signature is a reproducible element of a particularpolysaccharide being manipulated with a particular experimentalconstraint. For instance, some signatures of the HLGAGs which have beenidentified and demonstrated to be useful are ΔUH_(NAC,6S)GH_(NS,3S,6S),ΔUH_(NS,6S)GH_(NS,3S,6S); ΔUH_(NAC,6S)GH_(NS,3S); orΔUH_(NS,6S)GH_(NS,3S). When an HLGAG containing sample is subjected tocapillary electrophoresis following heparinase treatment this signaturewill be identified and is capable of being quantitated.

The signature component may be biologically active or inactive.Important information can be derived from the signature componentwhether it is an active component or an inactive component. A signaturewhich has biological activity is an oligosaccharide that is known toproduce a specific biological function. For instance thetetrasaccharides ΔUH_(NAC,6S)GH_(NS,3S,6S), ΔUH_(NS,6S)GH_(NS,3S,6S);ΔUH_(NAC,6S)GH_(NS,3S); or ΔUH_(NS,6S)GH_(NS,3S) of HLGAGs are known tobe part of the sequences possessing anti-coagulant activity resulting inthe inhibition of factor Xa. Thus the presence of this component in asample is directly indicative of the anti-coagulant activity of theHLGAG.

Signatures that have biological activity can be used for a variety ofpurposes. For instance, these types of signatures are useful formonitoring batch-to-batch variability of a polysaccharide preparation.The purity of each batch may be determined by determining the amount ofactive signature component in the batch. These signatures are alsouseful for monitoring the presence of active components in the sample,when the presence of the signature component in the sample is indicativeof an active component in the sample. For instance, the signaturecomponent may be used to follow the active component through aprocessing procedure. Using this method one can test the products aftereach separation step to determine which fraction contains thebiologically active component. The amount of active components in thesample can also be quantified by determining the amount of signaturecomponent in the sample.

The methods may also be performed on at least two samples to determinewhich sample has the most activity or to otherwise compare the purity ofthe samples. In this case the relative amounts of signature component ineach of the at least two samples is determined. The highest relativelevel of signature component is indicative of the most active sample.

Additionally, the active signature can be used to identify biologicallyactive molecules by screening compounds or libraries of compounds.Libraries include, for instance, phage display libraries, combinatoriallibraries, libraries of peptoids and non-peptide synthetic moieties.Phage display can be particularly effective in identifying peptideswhich interact with the signature components, including humanantibodies. Briefly, one prepares a phage library (using e.g. m13, fd,or lambda phage), displaying inserts from 4 to about 80 amino acidresidues using conventional procedures. The inserts may represent, forexample, a completely degenerate or biased array. One then can selectphage-bearing inserts which bind to the signature component. Thisprocess can be repeated through several cycles of reselection of phagethat bind to the signature component. Repeated rounds lead to enrichmentof phage bearing particular sequences. DNA sequence analysis can beconducted to identify the sequences of the expressed polypeptides. Theminimal linear portion of the sequence that binds to the signaturecomponent can be determined. One can repeat the procedure using a biasedlibrary containing inserts containing part or all of the minimal linearportion plus one or more additional degenerate residues upstream ordownstream thereof. Yeast two-hybrid screening methods also may be usedto identify polypeptides that bind to the signature component. Peptideand non-peptide libraries which are based on a known signature componentcan easily be generated by those of skill in the art. Commercialentities such as ArQule (Woburn, Mass.) prepare custom libraries for thegeneration of mimetic compounds.

Examples of biologically active portions of a polysaccharide include butare not limited to a tetrasaccharide of the AT-III biding domain ofheparin, a tetrasaccharide of the FGF biding domain of heparin,ΔUH_(NAC,6S)GH_(NS,3S,6S), ΔUH_(NS,6S)GH_(NS,3S,6S),ΔUH_(NAC,6S)GH_(NS,3S), or ΔUH_(NS,6S)GH_(NS,3S).

Signatures that are biologically inactive are oligosaccharides that arenot-associated with a specific known biological function. Theseoligosaccharides may have some biological function but not the specificfunction being analyzed. For instance, the oligosaccharide may actuallycause an inhibition of tumor cell growth, but not have any effect on thecoagulation cascade. If the polysaccharide sample is being evaluated forthe purpose of identifying the presence or amount of polysaccharideswhich are useful for anti-coagulation purposes, the oligosaccharidebeing detected is considered to be a biologically inactive signature.If, on the other hand, the polysaccharide sample is being evaluated forthe purpose of identifying the presence or amount of polysaccharideswhich are useful for preventing tumor cell proliferation, theoligosaccharide being detected is considered to be a biologically activesignature.

Signatures that are biologically inactive can be used for some of thesame purposes as biologically active signatures, as well as otherpurposes. Biologically inactive signatures can also be used to monitorbatch to batch variability of a polysaccharide preparation. Since twobatches are being compared to one another, both inactive and activesignatures can be used. Inactive signature components can also be usedfor monitoring the presence of active components in the sample when thepresence of the signature component in the sample is indicative of asample lacking a specific activity or having lower levels of thisactivity. Thus, if the presence of an inactive signature component isinversely proportional to the presence of an active component, then thepresence of the inactive component can provide important informationabout the activity of the sample. For instance if the inactive signaturecomponent is a degradation product of an active component of apolysaccharide, then the presence of the inactive component indicatesthat some of the active component has been broken down and thus thesample is less active than it would be if the inactive component werenot present.

An “experimental constraint” as used herein is a biochemical processperformed on a polysaccharide sample which results in a modification ofthe sample to allow the signature to be detected. Experimentalconstraints include but are not limited to separation methods, e.g.,mass spectrometry, capillary electrophoresis, high pressure liquidchromatography (HPLC), gel permeation chromatography, nuclear magneticresonance; enzymatic digestion, e.g., with an exoenzyme, an endoenzyme;chemical digestion; chemical modification; chemical peeling (i.e.,removal of a monosaccharide unit); and enzymatic modification, forinstance sulfation at a particular position with a heparan sulfatesulfotransferases.

The signature can be identified by any means which is consistent withthe experimental constraint used. Molecular weight of a signaturecomponent, for instance, may be determined by several methods includingmass spectrometry. The use of mass spectrometry for determining themolecular weight of polysaccharides is well known in the art. MassSpectrometry has been used as a powerful tool to characterizepolysaccharides because of its accuracy (±1 Dalton) in reporting themasses of fragments generated (e.g., by enzymatic cleavage), and alsobecause only pM sample concentrations are required. For example,matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS)has been described for identifying the molecular weight ofpolysaccharide fragments in publications such as Rhomberg, A. J. et al,PNAS, USA, v. 95, p. 4176-4181 (1998); Rhomberg, A. J. et al, PNAS, USA,v. 95, p. 12232-12237 (1998); and Ernst, S. et. al., PNAS, USA, v. 95,p. 4182-4187 (1998), each of which is hereby incorporated by reference.Other types of mass spectrometry known in the art, such as, electronspray-MS, fast atom bombardment mass spectrometry (FAB-MS) andcollision-activated dissociation mass spectrometry (CAD) can also beused to identify the molecular weight of the polysaccharide fragments.

The mass spectrometry data may be a valuable tool to ascertaininformation about the polysaccharide signature component alone or afterthe polysaccharide has undergone degradation with enzymes or chemicals.After a molecular weight of a polysaccharide is identified, it may becompared to molecular weights of other known polysaccharides (e.g.,using the methods of U.S. patent application Ser. Nos. 09/557,997 and09/558,137 filed on Apr. 24, 2000). As shown in these patentapplications, one technique for comparing molecular weights is togenerate a mass line and compare the molecular weight of the unknownpolysaccharide to the mass line to determine a subpopulation ofpolysaccharides which have the same molecular weight. A “mass line” isan information database, preferably in the form of a graph or chartwhich stores information for each possible type of polysaccharide havinga unique sequence based on the molecular weight of the polysaccharide.Because mass spectrometry data indicates the mass of a fragment to 1 Daaccuracy, a length may be assigned uniquely to a fragment by looking upa mass on the mass line. Further, it may be determined from the massline that, within a fragment of a particular length higher than adisaccharide, there is a minimum of 4.02 Da different in massesindicating that two acetate groups (84.08 Da) replaced a sulfate group(80.06 Da). Therefore, a number of sulfates and acetates of apolysaccharide fragment may be determined from the mass from the massspectrometry data and, such number may be assigned to the polysaccharidefragment.

In addition to molecular weight, other properties of a signaturecomponent may be determined. The compositional ratios of substituents orchemical units (quantity and type of total substituents or chemicalunits) may be determined using methodology known in the art, such ascapillary electrophoresis. A polysaccharide may be subjected to a firstexperimental constraint such as enzymatic or chemical degradation toseparate the polysaccharide into smaller fragments. These fragments thenmay be subjected to a second experimental constraint, that is, they maybe separated using capillary electrophoresis to determine the quantityand type of substituents or chemical units present in thepolysaccharide. Alternatively the polysaccharide may be subjected to asingle experimental constraint such as capillary electrophoreses,without prior enzymatic degradation.

In the method of capillary gel-electrophoresis, reaction samples may beanalyzed by small-diameter, gel-filled capillaries. The small diameterof the capillaries (50 μm) allows for efficient dissipation of heatgenerated during electrophoresis. Thus, high field strengths can be usedwithout excessive Joule heating (400 V/m), lowering the separation timeto about 20 minutes per reaction run, therefor increasing resolutionover conventional gel electrophoresis. Additionally, many capillariesmay be analyzed in parallel, allowing amplification of generatedpolysaccharide information.

Other methods for assessing the signature component may also beutilized. For instance, other methods include methods which rely onparameters such as viscosity (Jandik, K. A., Gu, K. and Linhardt, R. J.,(1994), Glycobiology, 4:284-296) or total UV absorbance (Ernst, S. etal., (1996), Biochem. J, 315:589-597).

HLGAG fragments may be degraded using enzymes such as heparin lyaseenzymes (heparinases) or nitrous acid and they may also be modifiedusing different enzymes that transfer sulfate groups to the positionsmentioned earlier or remove the sulfate groups from those positions. Themodifying enzymes are exolytic and non-processive which means that theyjust act once on the non reducing end and will let go of the heparinchain without sequentially modifying the rest of the chain. For each ofthe modifiable positions in the disaccharide unit there exits amodifying enzyme. An enzyme that adds a sulfate group is called asulfotransferase and an enzyme that removes a sulfate group is called asulfatase. The modifying enzymes include 2-O sulfatase/sulfotransferase,3-O sulfatase/sulfotransferase, 6-O sulfatase/sulfotransferase andN-deacetylase-N-sulfotransferase. The function of these enzymes isevident from their names, for example a 2-O sulfotransferase transfers asulfate group to the 2-O position of an iduronic acid (2-O sulfatedglucuronic acid is a rare occurrence in the HLGAG chains) and a 2-Osulfatase removes the sulfate group from the 2-O position of an iduronicacid.

HLGAG degrading enzymes include but are not limited to heparinase-I,heparinase-II , heparinase-III, D-glucuronidase and L-iduronidase,modified version so f heparinases, variants and functionally activefragments thereof. The three heparinases from Flavobacterium heparinumare enzymatic tools that have been used for the generation of LMWH(5,000-8,000 Da) and ultra-low molecular weight heparin (˜3,000 Da).Heparinase I cleaves highly sulfated regions of HLGAGs at 2-O sulfateduronic acids, whereas heparinase II has a broader substrate specificityand cleaves glycosidic linkages containing both 2-O sulfated andnonsulfated uronic acids (Ernst, S., Langer, R., Cooney, C. L. &Sasisekharan, R. (1995) Crit Rev Biochem Mol Biol 30, 387-444).Heparinase III, as opposed to heparinase I, cleaves primarilyundersulfated regions of HLGAGs, viz., glycosidic linkages containing anonsulfated uronic acid (Ernst, S., Langer, R., Cooney, C. L. &Sasisekharan, R. (1995) Crit Rev Biochem Mol Biol 30, 387-444). Multipleinvestigations into the substrate specificity of the heparinases hasincreased their usefulness as tools to develop structure-functionrelationships for HLGAGs. Several patents and patent applicationsdescribe useful modifications and variants and fragments of heparinase,including U.S. Pat. No. 6,217,863 B1 and pending applications Ser. Nos.09/384,959 and 09/802,285. Other modifications and variants are alsouseful. A more detailed understanding is required to maximize theirusefulness as generators of pharmacological LMWH. The discoveries of theinvention provide some more of this detail (as described below).

Glucuronidase and iduronidase, as their name suggests, cleave at theglycosidic linkage after a glucuronic acid and iduronic acidrespectively. Nitrous acid clips randomly at glycosidic linkages after aN-sulfated hexosamine and converts the six membered hexosamine ring to a5-membered anhydromannitol ring.

The methods for analysing polysaccharides by identifying the presence ofa signature component may be used to provide a qualitative assessment ofthe polysaccharide (e.g., whether the signature component is present orabsent) or a quantitative assessment (e.g., the amount of signaturecomponent present to indicate sample quality such as activity, purity orsimply to compare different samples). The method in some aspects isperformed by identifying a component within the polysaccharide sampleand determining a quantitative value of the amount of component. In someembodiments the method involves identifying and quantifying at least twocomponents.

The quantitative value may be calculated by any means, such as, bydetermining the area under the curve (AUC) when the sample is processedby capillary electrophoresis, the response factor (RF), or the percentrelative amount of each fraction present in the sample. Methods formaking these types of calculations are described below in detail in theExamples section. Briefly, the AUC can be calculated directly from a CEspectrum. The response factor is that amount of signature that gives thesame response as a control oligosaccharide. The RF can be calculated,for example, in terms of absorbance and compared with the absorbance ofa control sample. The percent relative amount of each fraction presentin the sample may be determined according to the following equation:PRA=RF×AUC_(%R)wherein

-   -   PRA=percent relative amount of each fraction    -   RF=response factor    -   AUC_(%R)=percent relative AUC [(100×AUC_(C))/AUC_(T))]    -   AUC_(C)=Area under the curve for one component    -   AUC_(T)=the sum of the Area under the curve for all components.

The data can be processed individually or by a computer. For instance, acomputer-implemented method for generating a data structure, tangiblyembodied in a computer-readable medium, representing a quantitativevalue of a component of a polysaccharide may be performed according tothe invention. The quantitative determination is made by performing theabove calculation.

A computer system that may implement the above as a computer programtypically may include a main unit connected to both an output devicewhich displays information to a user and an input device which receivesinput from a user. The main unit generally includes a processorconnected to a memory system via an interconnection mechanism. The inputdevice and output device also may be connected to the processor andmemory system via the interconnection mechanism.

One or more output devices may be connected to the computer system.Example output devices include a cathode ray tube (CRT) display, liquidcrystal displays (LCD), printers, communication devices such as a modem,and audio output. One or more input devices also may be connected to thecomputer system. Example input devices include a keyboard, keypad, trackball, mouse, pen and tablet, communication device, and data inputdevices such as sensors. The subject matter disclosed herein is notlimited to the particular input or output devices used in combinationwith the computer system or to those described herein.

The computer system may be a general purpose computer system which isprogrammable using a computer programming language, such as C++, Java,or other language, such as a scripting language or assembly language.The computer system also may include specially-programmed, specialpurpose hardware such as, for example, an Application-SpecificIntegrated Circuit (ASIC). In a general purpose computer system, theprocessor typically is a commercially-available processor, of which theseries x86, Celeron, and Pentium processors, available from Intel, andsimilar devices from AMD and Cyrix, the 680X0 series microprocessorsavailable from Motorola, the PowerPC microprocessor from IBM and theAlpha-series processors from Digital Equipment Corporation, areexamples. Many other processors are available. Such a microprocessorexecutes a program called an operating system, of which Windows NT,Linux, UNIX, DOS, VMS and OS8 are examples, which controls the executionof other computer programs and provides scheduling, debugging,input/output control, accounting, compilation, storage assignment, datamanagement and memory management, and communication control and relatedservices. The processor and operating system define a computer platformfor which application programs in high-level programming languages maybe written.

A memory system typically includes a computer readable and writeablenonvolatile recording medium, of which a magnetic disk, a flash memoryand tape are examples. The disk may be removable, such as a “floppydisk,” or permanent, known as a hard drive. A disk has a number oftracks in which signals are stored, typically in binary form, i.e., aform interpreted as a sequence of one and zeros. Such signals may definean application program to be executed by the microprocessor, orinformation stored on the disk to be processed by the applicationprogram. Typically, in operation, the processor causes data to be readfrom the nonvolatile recording medium into an integrated circuit memoryelement, which is typically a volatile, random access memory such as adynamic random access memory (DRAM) or static memory (SRAM). Theintegrated circuit memory element typically allows for faster access tothe information by the processor than does the disk. The processorgenerally manipulates the data within the integrated circuit memory andthen copies the data to the disk after processing is completed. Avariety of mechanisms are known for managing data movement between thedisk and the integrated circuit memory element, and the subject matterdisclosed herein is not limited to such mechanisms. Further, the subjectmatter disclosed herein is not limited to a particular memory system.

The subject matter disclosed herein is not limited to a particularcomputer platform, particular processor, or particular high-levelprogramming language. Additionally, the computer system may be amultiprocessor computer system or may include multiple computersconnected over a computer network. It should be understood that eachmodule (e.g. 110, 120) in FIG. 1 may be separate modules of a computerprogram, or may be separate computer programs. Such modules may beoperable on separate computers. Data (e.g., 104, 106, 110, 114 and 116)may be stored in a memory system or transmitted between computersystems. The subject matter disclosed herein is not limited to anyparticular implementation using software or hardware or firmware, or anycombination thereof. The various elements of the system, eitherindividually or in combination, may be implemented as a computer programproduct tangibly embodied in a machine-readable storage device forexecution by a computer processor. Various steps of the process may beperformed by a computer processor executing a program tangibly embodiedon a computer-readable medium to perform functions by operating on inputand generating output. Computer programming languages suitable forimplementing such a system include procedural programming languages,object-oriented programming languages, and combinations of the two.

Improved methods for preparing LMWH compositions were also discoveredaccording to the invention. The current methods of purifying lowmolecular weight heparin (LMWH) for clinical use include precipitationof a glycosaminoglycan mixture and the recovery of a fraction containingheparin fragments ranging in size from 1 to 14,000 Da. A standard methodutilized in the purification of heparin is described in N. Volpi,Biochemica t Biophysica Acta 1290 (1996) 299-307:

-   -   . . . slow moving and fast moving components of heparin were        purified as their barium salts at different temperatures, as        previously reported. Purified bovine intestinal mucosa heparin        was dissolved in water, and barium acetate (5%) was added slowly        with stirring (the pH of the solution was adjusted to 6.0-7.0).        After heating to 50°-70° C., the solution was left at room        temperature (20-25° C.) for 24 h. The precipitate obtained was        solubilized in water and transformed into its sodium salt on        Amberlite IR-120 resin. The crude slow moving heparin species        sodium salt was collected by precipitation with 2.0 volumes of        acetone and dried. The supernatant was maintained at 5° C. for        24 h and the precipitate was collected by centrifugation at        5° C. The fast moving species barium salt was purified as        reported for slow moving species.

The product obtained by this methodology is a heterogeneous mixture ofheparin fragments which have presented numerous difficulties whenadministered to patients due to the heterogeneous nature of the productas well as the lack of ability to quantify the levels of activecomponents in the mixture. In contrast, the novel purification strategydescribed herein, provides a substantially pure fraction of LMWH that isquantifiable and reproducible, and thus lacks many of the side effectsassociated with the prior art product. Surprisingly, it was discoveredaccording to the invention that the fraction referred to as the fastmoving component (the second fraction) which was discarded in the priorart methods actually has significant amounts of therapeutic activity.Thus, the method of the invention involves a similar type ofprecipitation reaction but involves isolation and manipulation of thepreviously discarded material.

In general the method of the invention involves a precipitation of HLGAGsample with a salt of divalent cations and weak anions. In someembodiments, the salt of the divalent cations and weak anions isselected from among the group including; barium, calcium, magnesium,strontium, copper, nickel, cadmium, zinc, mercury beryllium, nickel,palladium, platinum, iron, or tin. In some embodiments the salt ofdivalent cations and weak anions are acetates of cations of elements ofthe periodic table having divalent valence. In preferred embodiments thesalt of divalent cations and weak anions is barium acetate. In otherembodiments the salt of divalent cations and weak anions is calciumacetate or calcium chloride. In some embodiments other methods ofacetate precipitation which are known to those of skill in the art maybe used.

The precipitation may be performed in the temperature range from 0° C.to 70° C. The temperature for the precipitation may be 0° C., 2° C., 5°C., 10° C., 15° C., 20° C., 30° C., 40° C., 50° C., 60° C., or 70° C. ormay be any temperature within this range. In a preferred embodiment thetemperature of the precipitation is 4° C. In other embodiments, thetemperature of the precipitation will be room temperature, whichincludes temperatures in the range of 18° C. to 22° C.

The solvent used in the precipitation is a polar solvent. In someembodiments, the polar solvent is H₂O, ethanol, acetone, H₂O mixed withethanol, H₂O mixed with acetone, acetone. In some embodiments the polarsolvent has a volume to volume H2O:ethanol ratio in the range of: 99:1,95:5, 90:10, 85:15, 80:20, 75:25, 65:35, 55:45, 50:50, 45:55, 35:65,25:75, 20:80, 15:85, 10:90, 5:95, or 1:99. In other embodiments thepolar solvent has a volume to volume H₂O:acetone ratio in the range of99:1, 95:5, 90:10, 85:15, 80:20, 75:25, 65:35, 55:45, 50:50, 45:55,35:65, 25:75, 20:80, 15:85, 10:90, 5:95, or 1:99. In still otherembodiments the polar solvent is a mixture of H₂O, ethanol, and acetone.

Following the precipitation step the sample (second fraction) mayoptionally be subjected to an ion-exchange process prior to beingfurther processed. In some instances, the sample is passed through anion-exchange column, such as an amberlite IR-120 column. Use of thistype of column is useful for removing the salt used in the precipitationstep and replacing it with another such as sodium.

The glycosaminoglycan-containing sample is a sample at least a fractionof which is composed of glycosaminoglycans or HLGAGs. As discussed abovethe term glycosaminoglycan or HLGAG include but are not limited toheparin, heparin analogs, LMWH, biotechnological heparin, chemicallymodified heparin, or synthetic heparin.

The glycosaminoglycan can be fractionated into heparin of a specificsize by varying the conditions described herein for temperature,solvent, and enzyme. Examples of results of temperature variations,though not intending to be limiting, illustrate the variation in thecontent of precipitation fractions based on temperature. The use of 5%w/v barium acetate at 4° C. results in a second fraction that iscomprised of LMWH as defined by the FDA. This fraction has high activityfor anticoagulation, and low amount of sulfation (<70%). The fractionleft in the supernatant at 4° C. can be categorized as the ultra lowmolecular weight heparin. In contrast to the 4° C. precipitation, roomtemperature precipitation results in fraction one that contains higherMW heparin (MW: 10,000-14,000), higher amount of sulfation (>85%), andlower activity for anticoagulation than does the precipitation performedat 4° C.

Another nonlimiting example is the use of 5% calcium acetate ormagnesium acetate instead of 5% barium acetate. At 4° C. this changewill result in precipitating fraction one (high molecular weightheparin) while leaving fraction two (low molecular weight heparin) inthe supernatant. Fraction two can then be precipitated from thesupernatant by adding a polar solvent such as ethanol or acetone.

In general, the higher molecular weight and/or higher charge fractionwill precipitate at higher temperature, with a lower amount of polarsolvent such as ethanol or acetone. Decreasing the temperature, and/orincreasing the amount of polar solvent may result in the precipitationof the fraction with lower molecular weight, lower charge, and higheranticoagulation activity. The precipitation parameters may be alteredwithout undue experimentation by one of ordinary skill in the art.

Following the precipitation, the second fraction, the LMWH fraction, isprocessed to produce a concentrated LMWH preparation. The term“concentrated LMWH preparation” refers to a preparation which has beenaltered in one way or another from the second isolated fraction. Theprocessing step may involve a separation step or a purification stepsuch as a precipitation. The processing of the LMWH preparation mayfurther be accomplished by enzymatic or chemical digestion to yield theconcentrated LMWH preparation. In one embodiment the fraction isdigested and the enzyme used in the digestion is Heparinase III or afunctionally active variant or fragment thereof. The term heparinase isused generically to encompass functionally active variants and fragmentsthereof in addition to the native heparinases. Several patents andpatent applications describe useful modifications and variants andfragments of heparinase, including U.S. Pat. No. 6,217,863 B1 andpending application Ser. Nos. 09/384,959 and 09/802,285. Heparinase IIIcauses depolymerization of heparin. Depending upon the concentration ofheparinase III used, and the period for which it is used (partial vsexhaustive digestion), heparin of specific molecular weight, and/orcharge is obtained. For example, although not intended to be limiting,is that a partial digestion of heparin with 1 molar equivalent ofheparinase III would result in a fraction of higher molecular weight,and/or higher charge than would a reaction with a longer digestion time.Also, increasing the molar equivalence of heparinase III will result ina fraction with lower molecular weight and/or lower charge than if alower molar equivalence of heparinase is used. In some embodiments,Heparinase III concentrations and length of digestions can be used incombination with salt, temperature, and solvent composition, asdescribed herein, to obtain heparin of specific molecular weight, chargeand/or biological activity.

Alternatively, the second fraction may be chemically degraded to yieldthe concentrated LMWH preparation. In one embodiment the fraction ischemical degraded using a method selected from the group including butnot limited to: oxidative depolymerization with H₂O₂ or CU⁺ and H₂O₂,deaminative cleavage with isoamyl nitrite, or nitrous acid,β-eliminative cleavage with benzyl ester of heparin by alkalinetreatment or by heparinase.

The second fraction of the precipitation, which is referred to as fastmoving heparin in the Volpi reference, differs from the first fraction,which is referred to by Volpi reference as the slow moving heparin. Theaverage molecular weight of the second fraction is 8,000 Dalton and theaverage molecular weight of the first fraction is 14,000 Dalton. Inaddition, the second fraction is comprised of 100% LMWH with LMWHdefined as a heparin sub-species of average molecular weight of lessthan 8,000 Da, and in which at least 60% of the molecules have amolecular weight less than 8,000 Da. Using this definition, the firstfraction is 0% LMWH.

The invention also includes compositions of LMWH preparations. Thecomposition of LMWH is a mixture of various molecular weight molecules.As described above, the homogenous mixture contains fragments that canrange in molecular weight but have an average molecular weight of lessthan 8,000 D. A composition of LMWH of compounds having a molecularweight range from 4,000-6,000 Daltons, for instance, is a mixture ofvarious LMWH in which the average size ranges from 4,000 to 6,000 Da. Insome embodiments, the percentage of LMWH that is from 4,000 to 6,000 Dain the sample is 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of thecomponents in the sample.

In some aspects the composition is a LMWH preparation having an anti-Xaactivity of at least 150 IU/mg. In some embodiments the LMWH preparationhas an anti-factor Xa:anti-factor IIa activity ratio of greater than 1,2, 3, 4, or 5.

In other aspects the composition is a LMWH preparation having ananti-factor Xa:anti-factor IIa activity ratio of greater than 5. In yetother aspects the composition is LMWH preparation having at least 3.5%ΔUH_(NAC,6S)GH_(NS,3S,6S), ΔUH_(NS,6S)GH_(NS,3S,6S);ΔUH_(NAC,6S)GH_(NS,3S); or ΔUH_(NS,6S)GH_(NS,3S). In some embodimentsthe LMWH preparation has at least 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0,8.0, 9.0, 10.0, 15.0, or 20.0% ΔUH_(NAC,6S)GH_(NS,3S,6S),ΔUH_(NS,6S)GH_(NS,3S,6S); ΔUH_(NAC,6S)GH_(NS,3S); orΔUH_(NS,6S)GH_(NS,3S).

The LMWH compositions of the invention may optionally be formulated in apharmaceutically acceptable carrier. The compositions may further beformulated into specific delivery devices. Thus in some embodiments ofthe invention the compositions are specifically formulated forintravenous, subcutaneous, oral, aerosol, or other mucosal form ofdelivery. In some embodiments the compositions are formulated insustained release devices as described below.

One of ordinary skill in the art, in light of the present disclosure, isenabled to produce substantially pure preparations of LMWH compositions.The LMWH preparations are prepared from HLGAG sources. A “HLGAG source”as used herein refers to heparin like glycosaminoglycan compositionwhich can be manipulated to, produce LMWH using standard technology,including enzymatic degradation etc. As described above, HLGAGs includebut are not limited to isolated heparin, chemically modified heparin,biotechnology prepared heparin, synthetic heparin, heparan sulfate, andLMWH. Thus HLGAGs can be isolated from natural sources, prepared bydirect synthesis, mutagenesis, etc. The HLGAGs may in some embodimentsbe substantially pure. As used herein, the term “substantially pure”means that the polysaccharides are essentially free of other substancesto an extent practical and appropriate for their intended use. Inparticular, the polysaccharides are sufficiently pure and aresufficiently free from other biological constituents of their hostsenvironments so as to be useful in, for example, producingpharmaceutical preparations.

LMWH preparations as used herein are salts of sulfated GAGs having anaverage molecular weight (MW) of less than 8000 Da and for which atleast 60% of all molecules have a MW less than 8000 Da. By definitionLMWH preparations are produced from an HLGAG sample. The term LMWH doesnot encompass polysaccharides which are synthesized directly as LMWHs,such as SR90107A. SR90107A is a synthetic polysaccharide having amolecular weight of approximately 1500 Da. These types of compounds,which are prepared directly as low molecular weight compounds ratherthan being prepared from a source of HLGAGs are not considered to fallwithin the class of LMWH. The term LMWH does include, however, syntheticHLGAGs which are processed to produce LMWHs.

Several different methods have been used for the commercial preparationof LMWHs. Direct size fractionation has been used to prepare LMWH(Fraxiparin) on an experimental scale but its poor yield has generallynegated its use on an industrial scale. For industrial productionpurposes, a number of chemical or enzymatic processes have beenutilized. Chemical processes take advantage of a wide range of reactionssuch as partial nitrous acid depolymerization (Fragmin), oxidativecleavage with H₂O₂ (Normiflo and Fluxum), oxidative cleavage with Cu⁺⁺and H₂O₂, or by benzylation followed by β-elimination and alkalinehydrolysis (Enoxaparin). Enzymatic methods to generate LMWH usingpartial β-eliminative depolymerization by heparinase I (Logiparin) havealso been described.

The LMWHs produced according to the invention have improved functionalproperties over prior art LMWH preparations. One advantage of thecompositions of the invention is that the amount of anti-coagulantactivity can be altered for therapeutic purposes. Depending on thesubject being treated and/or the condition of the subject it may bedesirable to increase or decrease the anti-coagulant activity of thecompounds. For instance, if a subject is undergoing an acute clottingevent, it is often desirable to administer to the subject a LMWHpreparation having high anti-coagulant activity, such as one of thecompositions having an activity of at least 150 IU/mg. Other subjectsmay only be at risk of developing a thrombotic disorder. It is generallydesirable to administer to these subjects a LMWH preparation having alower anti-coagulant activity. The ability to identify the percentage ofuncleaved AT-binding region, such as the decasaccharide having structureΔU_(2S)H_(NS,6S)I_(2S)H_(NS,6S)I_(2S)H_(NS,6S)IH_(NAc,6S)GH_(NS,3S,6S),or one of the tetrasaccharides ΔUH_(NAC,6S)GH_(NS,3S,6S),ΔUH_(NS,6S)GH_(NS,3S,6S); ΔUH_(NAC,6S)GH_(NS,3S); orΔUH_(NS,6S)GH_(NS,3S) (or related compounds) in a sample allowscompositions to be formulated with specific amounts of uncleaved, intactAT-binding region This ability to prepare LMWH with known percentages ofintact AT-binding region provides a method to quantitate the activity oftherapeutic compositions of LMWH. Thus, the methods of the inventionenable one of skill in the art to prepare or identify an appropriatecomposition of LMWH, depending on the subject and the disorder beingtreated.

As used herein, the word “intact” means uncleaved and complete. The term“AT-binding regions” refers to a region of HLGAG that specificallyinteracts with AT-III. The AT-binding region, includes thedecasaccharide compound with the structure:ΔU_(2S)H_(NS,6S)I_(2S)H_(NS,6S)I_(2S)H_(NS,6S)IH_(NAc,6S)GH_(NS,3S,6S)and the tetrasaccharides ΔUH_(NAC,6S)GH_(NS,3S,6S),ΔUH_(NS,6S)GH_(NS,3S,6S); ΔUH_(NAC,6S)GH_(NS,3S); andΔUH_(NS,6S)GH_(NS,3S). In some embodiments, the LMWH preparation is acomposition wherein at least 20% of the polysaccharide sequence in thecomposition is intact AT-binding region. In other embodiments at least25%, 30%, 35%, 40%, 45%, 50%, or 55% of the polysaccharide sequence inthe composition is intact AT-binding region. As discussed above, theoptimal percentage of intact AT-10 in a composition for treatment willvary depending on the medical condition under treatment. A higher levelof activity may be desirable for patients in danger of blood-clotformation than in patients under treatment for cancer, in whichanticoagulant activity is not desirable.

In other aspects the composition is a LMWH preparation having >15%disulfated disaccharides, < than 75% trisulfated disaccharides, 3-5%monosulfated disaccharides, > than 2% 4-7 tetrasaccharide. The LMWHpreparation has an average MW<8,000 of which at least 60% of the chainshave a MW<8,000. LMWH preparations having these properties have ananti-XA activity of >150 IU/mg and an antiXa/IIA value of > than 1.5.Compositions having these properties can be prepared by the abovedescribed methods. The material referred to as the fraction 2 LMWHpreparation is a composition having these properties. The composition offraction 1 is a Heparin material having <15% disulfated disaccharides, >than 75% trisulfated disaccharides, 0-3% monosulfated disaccharides,0-2% 4-7 tetrasaccharide, and having an average MW of 8000-14000.Fraction 1 material has an anti-XA activity of <150 IU/mg and anantiXa/IIA value of < 2.

The amount of AT-binding region in the LMWH preparations can bemanipulated by a variety of experimental parameters. The methods of theinvention make it possible to control the amounts of AT-binding regionin a LMWH preparation by enabling the quality control of LMWHpreparations using the signature component, by providing an improvedisolation procedure which results in the isolation of a LMWH-richpreparation, and by providing new rules for the cleavage specificitiesof heparinases. The first two of these properties are discussed indetail above.

The role of heparinases in preparing LMWHs with intact AT-bindingregions has been described in the prior art. Specifically a publishedsequence that contained an intact AT-III binding site, was described asbeingΔU_(2S)H_(NS,6S)IH_(NAc,6S)GH_(NS,3S,6S)I_(2S)H_(NS,6S)I_(2S)H_(NS,6S)(Toida, T., Hileman, R. E., Smith, A. E., Vlahova, P. I. & Linhardt, R.J. (1996) J Biol Chem 271, 32040-7). Furthermore, tetrasaccharidescontaining 3-O sulfate have been shown in the prior art to beuncleavable by any of the heparinases (Yamada, S., Yoshida, K., Sugiura,M, Sugahara, K., Khoo, K. H., Morris, H. R. & Dell, A. (1993) J BiolChem 268, 4780-7.), suggesting that linkages with a 3-O sulfatedglucosamine are resistant to cleavage. Surprisingly, it was discoveredthat these prior art teachings were incorrect. In the Examples, weconclusively showed through a variety of physical chemical techniquesthat the actual structure of AT-10 isΔU_(2S)H_(NS,6S)I_(2S)H_(NS,6S)I_(2S)H_(NS,6S)IH_(NAc,6S)GH_(NS,3S,6S)and therefore does not contain an intact AT-III binding site. In lightof the reinterpretation of the AT-10 structure, we sought to rexaminethe action of heparinases I-III towards AT binding, 3-O sulfatecontaining oligosaccharides. Given that AT-10 (ultra-LMWH, MW=2769.3 Da)is derived from controlled heparinase I cleavage of heparin, we alsosought to examine the functional consequences of an oligosaccharide,using established bio-analytical techniques. Such an understanding ofboth heparinase action and functional consequences is required for theefficient, optimal generation of LMWH for clinical use.

Thus, as shown in the Examples, it has been discovered that heparinase Iand II actually cleave the AT-binding region of HLGAGs resulting in theloss of intact AT-binding region, whereas heparinase III does not.Furthermore, it has been discovered that glucosamine 3-0 sulfation atthe reducing end of a glycosidic linkage imparts resistance toheparinase I, II and III cleavage. Examination of the biological andpharmacological consequences of a heparin oligosaccharide that containsonly a partial AT-III binding site shows that such an oligosaccharidehas significant anti-Xa activity but lacks some of the functionalattributes of heparin-like glycosaminoglycan containing an intact AT-IIIsite. Thus if a preparation of HLGAGs is produced, such as, for example,by the precipitation method described above, the preparation can befurther modified such that it has a higher or lower anti-coagulantactivity by enzymatically cleaving with heparinases. If the preparationis subjected to enzymatic cleavage by heparinase I and/or II theanti-coagulant activity will be reduced. If the preparation is treatedwith heparinase III the anti-coagulant activity will be enhanced.

These methods are also true for a broader class of compounds. Theteachings of the invention can be used to develop specializedpolysaccharide therapeutics from a wide variety of polysaccharidestarting materials. Once an active component is identified in apolysaccharide, that active component can be used as a signature for thequality control of the sample, and can be used to generate and identifytherapeutic compositions which are enhanced for a particular therapeuticactivity, and which have had the regions which are responsible for sideeffects removed.

The compositions may be administered therapeutically to a subject. Asused herein, a subject is a human, non-human primate, cow, horse, pigsheep, goat dog, cat, or rodent.

HLGAGs and LMWHs in particular have many therapeutic utilities. The LMWHcompositions of the invention can be used for the treatment of any typeof condition in which LMWH therapy has been identified as a usefultherapy. Thus, the invention is useful in a variety of in vitro, in vivoand ex vivo methods in which LMWH therapies are useful. For instance, itis known that LMWH compositions are useful for preventing coagulation,inhibiting cancer cell growth and metastasis, preventing angiogenesis,preventing neovascularization, preventing psoriasis. The LMWHcompositions may also be used in in vivo assays, such as a qualitycontrol sample.

Each of these disorders is well-known in the art and is described, forinstance, in Harrison's Principles of Internal Medicine (McGraw Hill,Inc., New York), which is incorporated by reference.

Thus, the LMWH preparations are useful for treating or preventingdisorders associated with coagulation. A “disease associated withcoagulation” as used herein refers to a condition characterized by localinflammation resulting from an interruption in the blood supply to atissue due to a blockage of the blood vessel responsible for supplyingblood to the tissue such as is seen for myocardial or cerebralinfarction. A cerebral ischemic attack or cerebral ischemia is a form ofischemic condition in which the blood supply to the brain is blocked.This interruption in the blood supply to the brain may result from avariety of causes, including an intrinsic blockage or occlusion of theblood vessel itself, a remotely originated source of occlusion,decreased perfusion pressure or increased blood viscosity resulting ininadequate cerebral blood flow, or a ruptured blood vessel in thesubarachnoid space or intracerebral tissue.

The methods of the invention are useful also for treating cerebralischemia. Cerebral ischemia may result in either transient or permanentdeficits and the seriousness of the neurological damage in a patient whohas experienced cerebral ischemia depends on the intensity and durationof the ischemic event. A transient ischemic attack is one in which theblood flow to the brain is interrupted only briefly and causes temporaryneurological deficits, which often are clear in less than 24 hours.Symptoms of TIA include numbness or weakness of face or limbs, loss ofthe ability to speak clearly and/or to understand the speech of others,a loss of vision or dimness of vision, and a feeling of dizziness.Permanent cerebral ischemic attacks, also called stroke, are caused by alonger interruption in blood flow to the brain resulting from either athromboembolism. A stroke causes a loss of neurons typically resultingin a neurologic deficit that may improve but that does not entirelyresolve. Thromboembolic stroke is due to the occlusion of anextracranial or intracranial blood vessel by a thrombus or embolus.Because it is often difficult to discern whether a stroke is caused by athrombosis or an embolism, the term “thromboembolism” is used to coverstrokes caused by either of these mechanisms.

The methods of the invention in some embodiments are directed to thetreatment of acute thromboembolic stroke using LMWHs. An acute stroke isa medical syndrome involving neurological injury resulting from anischemic event, which is an interruption in the blood supply to thebrain.

An effective amount of a LMWH preparation alone or in combination withanother therapeutic for the treatment of stroke is that amountsufficient to reduce in vivo brain injury resulting from the stroke. Areduction of brain injury is any prevention of injury to the brain whichotherwise would have occurred in a subject experiencing a thromboembolicstroke absent the treatment of the invention. Several physiologicalparameters may be used to assess reduction of brain injury, includingsmaller infarct size, improved regional cerebral blood flow, anddecreased intracranial pressure, for example, as compared topretreatment patient parameters, untreated stroke patients or strokepatients treated with thrombolytic agents alone.

The pharmaceutical LMWH preparation may be used alone or in combinationwith a therapeutic agent for treating a disease associated withcoagulation. Examples of therapeutics useful in the treatment ofdiseases associated with coagulation include anticoagulation agents,antiplatelet agents, and thrombolytic agents.

Anticoagulation agents prevent the coagulation of blood components andthus prevent clot formation. Anticoagulants include, but are not limitedto, heparin, warfarin, coumadin, dicumarol, phenprocoumon,acenocoumarol, ethyl biscoumacetate, and indandione derivatives.

Antiplatelet agents inhibit platelet aggregation and are often used toprevent thromboembolic stroke in patients who have experienced atransient ischemic attack or stroke. Antiplatelet agents include, butare not limited to, aspirin, thienopyridine derivatives such asticlopodine and clopidogrel, dipyridamole and sulfinpyrazone, as well asRGD mimetics and also antithrombin agents such as, but not limited to,hirudin.

Thrombolytic agents lyse clots which cause the thromboembolic stroke.Thrombolytic agents have been used in the treatment of acute venousthromboembolism and pulmonary emboli and are well known in the art (e.g.see Hennekens et al, J Am Coll Cardiol; v. 25 (7 supp), p. 18S-22S(1995); Holmes, et al, J Am Coll Cardiol; v.25 (7 suppl), p.10S-17S(1995)). Thrombolytic agents include, but are not limited to,plasminogen, a₂-antiplasmin, streptokinase, antistreplase, tissueplasminogen activator (tPA), and urokinase. “tPA” as used hereinincludes native tPA and recombinant tPA, as well as modified forms oftPA that retain the enzymatic or fibrinolytic activities of native tPA.The enzymatic activity of tPA can be measured by assessing the abilityof the molecule to convert plasminogen to plasmin. The fibrinolyticactivity of tPA may be determined by any in vitro clot lysis activityknown in the art, such as the purified clot lysis assay described byCarlson, et. al., Anal. Biochem. 168, 428-435 (1988) and its modifiedform described by Bennett, W. F. Et al., 1991, Supra, the entirecontents of which are hereby incorporated by reference.

In one embodiment the LMWH preparations are used for inhibitingangiogenesis. An effective amount for inhibiting angiogenesis of theLMWH preparation is administered to a subject in need of treatmentthereof. Angiogenesis as used herein is the inappropriate formation ofnew blood vessels. “Angiogenesis” often occurs in tumors whenendothelial cells secrete a group of growth factors that are mitogenicfor endothelium causing the elongation and proliferation of endothelialcells which results in a generation of new blood vessels. Several of theangiogenic mitogens are heparin binding peptides which are related toendothelial cell growth factors. The inhibition of angiogenesis cancause tumor regression in animal models, suggesting a use as atherapeutic anticancer agent. An effective amount for inhibitingangiogenesis is an amount of LMWH preparation which is sufficient todiminish the number of blood vessels growing into a tumor. This amountcan be assessed in an animal model of tumors and angiogenesis, many ofwhich are known in the art.

The LMWH preparations are also useful for inhibiting neovascularizationassociated with eye disease. In another embodiment, the LMWH preparationis administered to treat psoriasis. Psoriasis is a common dermatologicdisease causes by chronic inflammation.

LMWH containing compositions, may also inhibit cancer cell growth andmetastasis. Thus the methods of the invention are useful for treatingand/or preventing tumor cell proliferation or metastasis in a subject.The terms “prevent” and “preventing” as used herein refer to inhibitingcompletely or partially the biological effect, e.g., angiogenesis orproliferation or metastasis of a cancer or tumor cell, as well asinhibiting any increase in the biological effect, e.g., angiogenesis orproliferation or metastasis of a cancer or tumor cell.

The cancer may be a malignant or non-malignant cancer. Cancers or tumorsinclude but are not limited to biliary tract cancer; brain cancer;breast cancer; cervical cancer; choriocarcinoma; colon cancer;endometrial cancer; esophageal cancer; gastric cancer; intraepithelialneoplasms; lymphomas; liver cancer; lung cancer (e.g. small cell andnon-small cell); melanoma; neuroblastomas; oral cancer; ovarian cancer;pancreas cancer; prostate cancer; rectal cancer; sarcomas; skin cancer;testicular cancer; thyroid cancer; and renal cancer, as well as othercarcinomas and sarcomas.

A subject in need of treatment may be a subject who has a highprobability of developing cancer. These subjects include, for instance,subjects having a genetic abnormality, the presence of which has beendemonstrated to have a correlative relation to a higher likelihood ofdeveloping a cancer and subjects exposed to cancer-causing agents suchas tobacco, asbestos, or other chemical toxins, or a subject who haspreviously been treated for cancer and is in apparent remission.

Effective amounts of the composition containing LMWH of the inventionare administered to subjects in need of such treatment. Effectiveamounts are those amounts which will result in a desired reduction incellular proliferation or metastasis or prevent coagulation withoutcausing other medically unacceptable side effects. Such amounts can bedetermined with no more than routine experimentation. It is believedthat doses ranging from 1 nanogram/kilogram to 100 milligrams/kilogram,depending upon the mode of administration, will be effective. Theeffective percentage of intact LMWH may be determined with no more thanroutine experimentation. The absolute amount will depend upon a varietyof factors (including whether the administration is in conjunction withother methods of treatment, the number of doses and individual patientparameters including age, physical condition, size and weight) and canbe determined with routine experimentation. It is preferred generallythat a maximum dose be used, that is, the highest safe dose according tosound medical judgment. The mode of administration may be any medicallyacceptable mode including inhalation, oral, subcutaneous, intravenous,etc.

In some aspects of the invention the effective amount of a compositioncontaining LMWH is that amount effective to prevent invasion of a tumorcell across a barrier. The invasion and h6metastasis of cancer is acomplex process which involves changes in cell adhesion properties whichallow a transformed cell to invade and migrate through the extracellularmatrix (ECM) and acquire anchorage-independent growth properties.Liotta, L. A., et al., Cell 64:327-336 (1991). Some of these changesoccur at focal adhesions, which are cell/ECM contact points containingmembrane-associated, cytoskeletal, and intracellular signalingmolecules. Metastatic disease occurs when the disseminated foci of tumorcells seed a tissue which supports their growth and propagation, andthis secondary spread of tumor cells is responsible for the morbidityand mortality associated with the majority of cancers. Thus the term“metastasis” as used herein refers to the invasion and migration oftumor cells away from the primary tumor site.

The barrier for the tumor cells may be an artificial barrier in vitro ora natural barrier in vivo. In vitro barriers include but are not limitedto extracellular matrix coated membranes, such as Matrigel. Thus theLMWH compositions can be tested for their ability to inhibit tumor cellinvasion in a Matrigel invasion assay system as described in detail byParish, C. R., et al., “A Basement-Membrane Permeability Assay whichCorrelates with the Metastatic Potential of Tumour Cells,” Int. J.Cancer (1992) 52:378-383. Matrigel is a reconstituted basement membranecontaining type IV collagen, laminin, heparan sulfate proteoglycans suchas perlecan, which bind to and localize bFGF, vitronectin as well astransforming growth factor (TGF), urokinase-type plasminogen activator(uPA), tissue plasminogen activator (tPA), and the serpin known asplasminogen activator inhibitor type 1 (PAI-1). Other in vitro and invivo assays for metastasis have been described in the prior art, see,e.g., U.S. Pat. No. 5,935,850, issued on Aug. 10, 1999, which isincorporated by reference. An in vivo barrier refers to a cellularbarrier present in the body of a subject.

According to another aspect of the invention, there is provided methodsfor treating subjects in need of depletion of circulating heparin.Effective amounts of combinations of heparinases I, II, and III (ormodified forms thereof are utilized) in this aspect. For example,subjects undergoing open heart surgery or hemodialysis often are in needof depletion of medically undesirable amounts of heparin in blood as aresult of the surgery or hemodialysis. By using a combination ofheparinase I or II and heparinase III the appropriate amount oftherapeutically active (anti-coagulant function) can be administered toa subject to obtain an appropriate balance of the coagulation cascade.Effective amounts of the combination of heparinases are those amountswhich will result in a desired reduction in circulating heparin levelswithout complete depletion and without causing any other medicallyunacceptable side effects.

In general the therapeutically useful amounts of the combination ofheparinases can be determined with no more than routine experimentation.It is believed that doses ranging from I nanogram/kilogram to 100milligrams/kilogram, depending upon the mode of administration, will beeffective. The absolute amount will depend upon a variety of factors(including whether the administration is in conjunction with othermethods of treatment, the number of doses and individual patientparameters including age, physical condition, size and weight) and canbe determined with routine experimentation. It is preferred generallythat a maximum dose be used, that is, the highest safe dose according tosound medical judgment. The mode of administration may be any medicallyacceptable mode including oral, subcutaneous, intravenous, etc.

In general, when administered for therapeutic purposes, the formulationsof the invention are applied in pharmaceutically acceptable solutions.Such preparations may routinely contain pharmaceutically acceptableconcentrations of salt, buffering agents, preservatives, compatiblecarriers, adjuvants, and optionally other therapeutic ingredients.

The compositions of the invention may be administered per se (neat) orin the form of a pharmaceutically acceptable salt. When used in medicinethe salts should be pharmaceutically acceptable, butnon-pharmaceutically acceptable salts may conveniently be used toprepare pharmaceutically acceptable salts thereof and are not excludedfrom the scope of the invention. Such pharmacologically andpharmaceutically acceptable salts include, but are not limited to, thoseprepared from the following acids: hydrochloric, hydrobromic, sulphuric,nitric, phosphoric, maleic, acetic, salicylic, p-toluene sulphonic,tartaric, citric, methane sulphonic, formic, malonic, succinic,naphthalene-2-sulphonic, and benzene sulphonic. Also, pharmaceuticallyacceptable salts can be prepared as alkaline metal or alkaline earthsalts, such as sodium, potassium or calcium salts of the carboxylic acidgroup.

Suitable buffering agents include: acetic acid and a salt (1-2% W/V);citric acid and a salt (1-3% W/V); boric acid and a salt (0.5-2.5% W/V);and phosphoric acid and a salt (0.8-2% W/V). Suitable preservativesinclude benzalkonium chloride (0.003-0.03% W/V); chlorobutanol (0.3-0.9%W/V); parabens (0.01-0.25% W/V) and thimerosal (0.004-0.02% W/V).

The present invention provides pharmaceutical compositions, for medicaluse, which comprise LMWH preparations together with one or morepharmaceutically acceptable carriers and optionally other therapeuticingredients. The term “pharmaceutically-acceptable carrier” as usedherein, and described more fully below, means one or more compatiblesolid or liquid filler, dilutants or encapsulating substances which aresuitable for administration to a human or other animal. In the presentinvention, the term “carrier” denotes an organic or inorganicingredient, natural or synthetic, with which the active ingredient iscombined to facilitate the application. The components of thepharmaceutical compositions also are capable of being commingled withthe LMWH of the present invention, and with each other, in a manner suchthat there is no interaction which would substantially impair thedesired pharmaceutical efficiency.

Compositions suitable for parenteral administration convenientlycomprise a sterile aqueous preparation of the polysaccharide, which canbe isotonic with the blood of the recipient. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solution,and isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose any bland fixed oil may be employed including synthetic mono- ordiglycerides. In addition, fatty acids such as oleic acid find use inthe preparation of injectables. Carrier formulations suitable forsubcutaneous, intramuscular, intraperitoneal, intravenous, etc.administrations may be found in Remington's Pharmaceutical Sciences,Mack Publishing Company, Easton, Pa.

A variety of administration routes are available. The particular modeselected will depend, of course, upon the particular percentage of LMWHselected, the particular condition being treated, and the dosagerequired for therapeutic efficacy. The methods of this invention,generally speaking, may be practiced using any mode of administrationthat is medically acceptable, meaning any mode that produces effectivelevels of an biological effect without causing clinically unacceptableadverse effects.

For use in therapy, an effective amount of the LMWH preparation can beadministered to a subject by any mode that delivers the LMWH to thedesired surface, e.g., mucosal, systemic. “Administering” thepharmaceutical composition of the present invention may be accomplishedby any means known to the skilled artisan. Preferred routes ofadministration include, but are not limited to, oral, parenteral,intramuscular, intranasal, intratracheal, inhalation, ocular, vaginaland rectal.

For oral administration, the compounds (i.e., LMWH preparations) can beformulated readily by combining the active compound(s) withpharmaceutically acceptable carriers well-known in the art. Suchcarriers enable the compounds of the invention to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions and the like, for oral ingestion by a subject to be treated.Pharmaceutical preparations for oral use can be obtained as solidexcipient, optionally grinding a resulting mixture, and processing themixture of granules, after adding suitable auxiliaries, if desired, toobtain tablets or dragee cores. Suitable excipients are, in particular,fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; cellulose preparations such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired,disintegrating agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodiumalginate. Optionally, the oral formulations may also be formulated insaline or buffers for neutralizing internal acid conditions or may beadministered without any carriers.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. Microspheres formulatedfor oral administration may also be used. Such microspheres have beenwell defined in the art. All formulations for oral administration shouldbe in dosages suitable for such administration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention may be conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebulizer, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol, the dosageunit may be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g. gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The compounds, when it is desirable to deliver them systemically, may beformulated for parenteral administration by injection, e.g., by bolusinjection or continuous infusion. Formulations for injection may bepresented in unit dosage form, e.g., in ampoules or in multi-dosecontainers, with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

Alternatively, the active compounds may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The compounds may also be formulated in rectal or vaginal compositionssuch as suppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long-acting formulationsmay be formulated with suitable polymeric or hydrophobic materials (forexample, as an emulsion in an acceptable oil) or ion exchange resins, oras sparingly soluble derivatives, for example, as a sparingly solublesalt.

The pharmaceutical compositions also may comprise suitable solid or gelphase carriers or excipients. Examples of such carriers or excipientsinclude, but are not limited to, calcium carbonate, calcium phosphate,various sugars, starches, cellulose derivatives, gelatin, and polymerssuch as polyethylene glycols.

Suitable liquid or solid pharmaceutical preparation forms are, forexample, aqueous or saline solutions for inhalation, microencapsulated,encochleated, coated onto microscopic gold particles, contained inliposomes, nebulized, aerosols, pellets for implantation into the skin,or dried onto a sharp object to be scratched into the skin. Thepharmaceutical compositions also include granules, powders, tablets,coated tablets, (micro) capsules, suppositories, syrups, emulsions,suspensions, creams, drops or preparations with protracted release ofactive compounds, in whose preparation excipients and additives and/orauxiliaries such as disintegrants, binders, coating agents, swellingagents, lubricants, flavorings, sweeteners or solubilizers arecustomarily used as described above. The pharmaceutical Compositions aresuitable for use in a variety of drug delivery systems. For a briefreview of methods for drug delivery, see Langer, Science 249:1527-1533,(1990), which is incorporated herein by reference.

The compositions may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.All methods include the step of bringing the active LMWH intoassociation with a carrier which constitutes one or more accessoryingredients. In general, the compositions are prepared by uniformly andintimately bringing the polysaccharide into association with a liquidcarrier, a finely divided solid carrier, or both, and then, ifnecessary, shaping the product. The polysaccharide may be storedlyophilized.

Other delivery systems can include time-release, delayed release orsustained release delivery systems. Such systems can avoid repeatedadministrations of the LMWH of the invention, increasing convenience tothe subject and the physician. Many types of release delivery systemsare available and known to those of ordinary skill in the art. Theyinclude polymer based systems such as polylactic and polyglycolic acid,polyanhydrides and polycaprolactone; nonpolymer systems that are lipidsincluding sterols such as cholesterol, cholesterol esters and fattyacids or neutral fats such as mono-, di and triglycerides; hydrogelrelease systems; silastic systems; peptide based systems; wax coatings,compressed tablets using conventional binders and excipients, partiallyfused implants and the like. Specific examples include, but are notlimited to: (a) erosional systems in which the polysaccharide iscontained in a form within a matrix, found in U.S. Pat. No. 4,452,775(Kent); U.S. Pat. No. 4,667,014 (Nestor et al.); and U.S. Pat. Nos.4,748,034 and 5,239,660 (Leonard) and (b) diffusional systems in whichan active component permeates at a controlled rate through a polymer,found in U.S. Pat. No. 3,832,253 (Higuchi et al.) and U.S. Pat. No.3,854,480 (Zaffaroni). In addition, a pump-based hardware deliverysystem can be used, some of which are adapted for implantation.

When administered to a patient undergoing cancer treatment, the LMWHcompositions may be administered in cocktails containing otheranti-cancer agents. The compositions may also be administered incocktails containing agents that treat the side-effects of radiationtherapy, such as anti-emetics, radiation protectants, etc.

Anti-cancer drugs that can be co-administered with the compounds of theinvention include, but are not limited to Acivicin; Aclarubicin;Acodazole Hydrochloride; Acronine; Adriamycin; Adozelesin; Aldesleukin;Altretamine; Ambomycin; Ametantrone Acetate; Aminoglutethimide;Amsacrine; Anastrozole; Anthramycin; Asparaginase; Asperlin;Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa; Bicalutamide;Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin; BleomycinSulfate; Brequinar Sodium; Bropirimine; Busulfan; Cactinomycin;Calusterone; Caracemide; Carbetimer; Carboplatin; Carmustine; CarubicinHydrochloride; Carzelesin; Cedefingol; Chlorambucil; Cirolemycin;Cisplatin; Cladribine; Crisnatol Mesylate; Cyclophosphamide; Cytarabine;Dacarbazine; Dactinomycin; Daunorubicin Hydrochloride; Decitabine;Dexormaplatin; Dezaguanine; Dezaguanine Mesylate; Diaziquone; Docetaxel;Doxorubicin; Doxorubicin Hydrochloride; Droloxifene; DroloxifeneCitrate; Dromostanolone Propionate; Duazomycin; Edatrexate; EflomithineHydrochloride; Elsamitrucin; Enloplatin; Enpromate; Epipropidine;Epirubicin Hydrochloride; Erbulozole; Esorubicin Hydrochloride;Estramustine; Estramustine Phosphate Sodium; Etanidazole; Etoposide;Etoposide Phosphate; Etoprine; Fadrozole Hydrochloride; Fazarabine;Fenretinide; Floxuridine; Fludarabine Phosphate; Fluorouracil;Flurocitabine; Fosquidone; Fostriecin Sodium; Gemcitabine; GemcitabineHydrochloride; Hydroxyurea; Idarubicin Hydrochloride; Ifosfamide;Ilmofosine; Interferon Alfa-2a; Interferon Alfa-2b; Interferon Alfa-n1;Interferon Alfa-n3; Interferon Beta-I a; Interferon Gamma-I b;Iproplatin; Irinotecan Hydrochloride; Lanreotide Acetate; Letrozole;Leuprolide Acetate; Liarozole Hydrochloride; Lometrexol Sodium;Lomustine; Losoxantrone Hydrochloride; Masoprocol; Maytansine;Mechlorethamine Hydrochloride; Megestrol Acetate; Melengestrol Acetate;Melphalan; Menogaril; Mercaptopurine; Methotrexate; Methotrexate Sodium;Metoprine; Meturedepa; Mitindomide; Mitocarcin; Mitocromin; Mitogillin;Mitomalcin; Mitomycin; Mitosper; Mitotane; Mitoxantrone Hydrochloride;Mycophenolic Acid; Nocodazole; Nogalamycin; Ormaplatin; Oxisuran;Paclitaxel; Pegaspargase; Peliomycin; Pentamustine; Peplomycin Sulfate;Perfosfamide; Pipobroman; Piposulfan; Piroxantrone Hydrochloride;Plicamycin; Plomestane; Porfimer Sodium; Porfiromycin; Prednimustine;Procarbazine Hydrochloride; Puromycin; Puromycin Hydrochloride;Pyrazofurin; Riboprine; Rogletimide; Safingol; Safingol Hydrochloride;Semustine; Simtrazene; Sparfosate Sodium; Sparsomycin; SpirogermaniumHydrochloride; Spiromustine; Spiroplatin; Streptonigrin; Streptozocin;Sulofenur; Talisomycin; Tecogalan Sodium; Tegafur; TeloxantroneHydrochloride; Temoporfin; Teniposide; Teroxirone; Testolactone;Thiamiprine; Thioguanine; Thiotepa; Tiazofurin; Tirapazamine; TopotecanHydrochloride; Toremifene Citrate; Trestolone Acetate; TriciribinePhosphate; Trimetrexate; Trimetrexate Glucuronate; Triptorelin;Tubulozole Hydrochloride; Uracil Mustard; Uredepa; Vapreotide;Verteporfin; Vinblastine Sulfate; Vincristine Sulfate; Vindesine;Vindesine Sulfate; Vinepidine Sulfate; Vinglycinate Sulfate;Vinleurosine Sulfate; Vinorelbine Tartrate; Vinrosidine Sulfate;Vinzolidine Sulfate; Vorozole; Zeniplatin; Zinostatin; ZorubicinHydrochloride.

The LMWH compositions may also be linked to a targeting molecule. Atargeting molecule is any molecule or compound which is specific for aparticular cell or tissue and which can be used to direct the LMWH tothe cell or tissue. Preferably the targeting molecule is a moleculewhich specifically interacts with a cancer cell or a tumor. Forinstance, the targeting molecule may be a protein or other type ofmolecule that recognizes and specifically interacts with a tumorantigen.

Tumor-antigens include Melan-A/MART-1, Dipeptidyl peptidase IV (DPPIV),adenosine deaminase-binding protein (ADAbp), cyclophilin b, Colorectalassociated antigen (CRC)—C017-1A/GA733, Carcinoembryonic Antigen (CEA)and its immunogenic epitopes CAP-1 and CAP-2, etv6, aml1, ProstateSpecific Antigen (PSA) and its immunogenic epitopes PSA-1, PSA-2, andPSA-3, prostate-specific membrane antigen (PSMA), T-cellreceptor/CD3-zeta chain, MAGE-family of tumor antigens (e.g., MAGE-A1,MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9,MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3),MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5),GAGE-family of tumor antigens (e.g., GAGE-1, GAGE-2, GAGE-3, GAGE-4,GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V,MUM-1, CDK4, tyrosinase, p53, MUC family, HER2/neu, p21ras, RCAS1,fetoprotein, E-cadherin, catenin, , p120ctn, gp100^(Pmel117), PRAME,NY-ESO-1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40),SSX-1, SSX-4, SSX-5, SCP-1, CT-7, cdc27, adenomatous polyposis coliprotein (APC), fodrin, P1A, Connexin 37, Ig-idiotype, p15, gp75, GM2 andGD2 gangliosides, viral products such as human papilloma virus proteins,Smad family of tumor antigens, lmp-1, EBV-encoded nuclear antigen(EBNA)-1, and c-erbB-2.

Examples of tumor antigens which bind to either or both MHC class I andMHC class II molecules, see the following references: Coulie, Stem Cells13:393-403, 1995; Traversari et al., J. Exp. Med. 176:1453-1457, 1992;Chaux et al., J. Immunol. 163:2928-2936, 1999; Fujie et al., Int. J.Cancer 80:169-172, 1999; Tanzarella et al., Cancer Res. 59:2668-2674,1999; van der Bruggen et al., Eur. J. Immunol. 24:2134-2140, 1994; Chauxet al., J. Exp. Med. 189:767-778, 1999; Kawashima et al, Hum. Immunol.59:1-14, 1998; Tahara et al., Clin. Cancer Res. 5:2236-2241, 1999;Gaugler et al., J. Exp. Med. 179:921-930, 1994; van der Bruggen et al.,Eur. J. Immunol. 24:3038-3043, 1994; Tanaka et al., Cancer Res.57:4465-4468, 1997; Oiso et al., Int. J. Cancer 81:387-394, 1999; Hermanet al., Immunogenetics 43:377-383, 1996; Manici et al., J. Exp. Med.189:871-876, 1999; Duffour et al., Eur. J. Immunol. 29:3329-3337, 1999;Zom et al., Eur. J. Immunol. 29:602-607, 1999; Huang et al., J.Immunol.162:6849-6854, 1999; Böel et al., Immunity 2:167-175, 1995; Vanden Eynde et al., J. Exp. Med. 182:689-698, 1995; De Backer et al.,Cancer Res. 59:3157-3165, 1999; Jäger et al., J. Exp. Med. 187:265-270,1998; Wang et al., J. Immunol. 161:3596-3606, 1998; Aarnoudse et al.,Int. J. Cancer 82:442-448, 1999; Guillouxetal., J. Exp. Med.183:1173-1183, 1996;Lupetti et al., J. Exp. Med. 188:1005-1016, 1998;Wölfel et al., Eur. J. Immunol. 24:759-764, 1994; Skipper et al., J.Exp. Med. 183:527-534, 1996; Kang et al., J. Immunol. 155:1343-1348,1995; Morel et al., Int. J. Cancer 83:755-759, 1999; Brichard et al.,Eur. J. Immunol. 26:224-230, 1996; Kittlesen et al., J. Immunol.160:2099-2106, 1998; Kawakami et al., J. Immunol. 161:6985-6992, 1998;Topalian et al., J. Exp. Med. 183:1965-1971, 1996; Kobayashi et al.,Cancer Research 58:296-301, 1998; Kawakami et al., J. Immunol.154:3961-3968, 1995; Tsai et al., J. Immunol. 158:1796-1802, 1997; Coxet al., Science 264:716-719, 1994; Kawakami et al., Proc. Natl. Acad.Sci. USA 91:6458-6462, 1994; Skipper et al., J. Immunol. 157:5027-5033,1996; Robbins et al., J. Immunol. 159:303-308, 1997;.Castelli et al, J.Immunol. 162:1739-1748, 1999; Kawakami et al., J. Exp. Med. 180:347-352,1994; Castelli et al., J. Exp. Med. 181:363-368, 1995; Schneider et al.,Int. J. Cancer 75:451-458, 1998; Wang et al., J. Exp. Med.183:1131-1140, 1996;Wang et al., J. Exp. Med. 184:2207-2216, 1996;Parkhurst et al., Cancer Research 58:4895-4901, 1998; Tsang et al., J.Natl Cancer Inst 87:982-990, 1995; Correale et al., J. Natl Cancer Inst89:293-300, 1997; Coulie et al., Proc. Natl. Acad. Sci. USA92:7976-7980, 1995; Wölfel et al., Science 269:1281-1284, 1995; Robbinset al., J. Exp. Med. 183:1185-1192, 1996; Brändle et al., J. Exp. Med.183:2501-2508, 1996; ten Bosch et al., Blood 88:3522-3527, 1996;Mandruzzato et al., J. Exp. Med. 186:785-793, 1997; Guéguen et al., J.Immunol. 160:6188-6194, 1998; Gjertsen et al., Int. J. Cancer72:784-790, 1997; Gaudin et al., J. Immunol. 162:1730-1738, 1999; Chiariet al., Cancer Res. 59:5785-5792, 1999; Hogan et al., Cancer Res.58:5144-5150, 1998; Pieper et al., J. Exp. Med. 189:757-765, 1999; Wangetal., Science 284:1351-1354, 1999; Fisk et al., J. Exp. Med.181:2109-2117, 1995; Brossart et al., Cancer Res. 58:732-736, 1998;Röpke et al., Proc. Natl. Acad. Sci. USA 93:14704-14707, 1996; Ikeda etal., Immunity 6:199-208, 1997; Ronsin et al., J. Immunol. 163:483-490,1999; Vonderheide et al., Immunity 10:673-679, 1999. These antigens aswell as others are disclosed in PCT Application PCT/US98/18601.

The following description of experiments performed is exemplary andnon-limiting to the scope of the claimed invention.

EXAMPLES Example 1 Sequencing of 3-O Sulfate Containing Decasaccharideswith a Partial Antithrombin III Binding Site

Introduction:

Heparin and heparan sulfate glycosaminoglycans represent an importantclass of molecules that interact with and modulate the activity ofgrowth factors, enzymes, and morphogens. Of the many biologicalfunctions for this class of molecules, one of its most importantfunctions is its interaction with antithrombin III (AT-III). AT-IIIbinding to a specific heparin pentasaccharide sequence, containing anunusual 3-O sulfate on a N-sulfated, 6-O sulfated glucosamine, increases1000-fold AT-III's ability to inhibit specific proteases in thecoagulation cascade. In this manner, heparin-like glycosaminoglycans(HLGAGs) play an important biological and pharmacological role in themodulation of blood clotting. Recently, a sequencing methodology wasdeveloped (U.S. patent application Ser. Nos. 09/557,997 and 09/558,137filed on Apr. 24, 2000, having common inventorship, which areincorporated by reference and Venkataraman, G., Shriver, Z., Raman, R. &Sasisekharan, R. (1999) Science 286, 537-42.) to furtherstructure-function relationships of this important class of molecules.This methodology combines a property-encoded nomenclature scheme (PEN),to handle the large information content (properties) of HLGAGs, withmatrix-assisted laser desorption ionization mass spectrometry (MALDI-MS)and enzymatic and chemical degradation as experimental constraints torapidly sequence picomole quantities of HLGAG oligosaccharides. Usingthe above PEN-MALDI approach, we found that the sequence of thedecasaccharide used in this study isΔU_(2S)H_(NS,6S)I_(2S)H_(NS,6S)I_(2S)H_(NS,6S)IH_(NAc,6S)GH_(NS,3S,6S)(±DDD4-7).We confirmed our results using Integral Glycan Sequencing and onedimensional proton nuclear magnetic resonance. Furthermore, we showedthat this approach is flexible and is able to derive sequenceinformation from an oligosaccharide mixture. Thus, this methodologymakes possible both the analysis of other unusual sequences inpolysaccharides such as heparin/heparan sulfate with importantbiological activities as well as provides the basis for the structuralanalysis of these pharmacologically important group of heparin/heparansulfates.

Methods

Abbreviations: HLGAG, heparin-like glycosaminoglycans; AT-III,antithrombin III; AT-10, AT-III fractionated decasaccharide isolatedfrom partial digestion of heparin; IGS, Integral Glycan Sequencing; PEN,property encoded nomenclature; MALDI-MS, matrix assisted laserdesorption ionization mass spectrometry; CE, capillary electrophoresis;HLGAG sequence abbreviations as follows, I, α-L-iduronic acid; G,β-D-glucuronic acid; ΔU, a Δ^(4.5) uronic acid; 2S, 3S, and 6S, 2-O,3-O, or 6-O sulfation respectively; NS and NAc, N-sulfation andN-acetylation of the glucosamine.

Materials. The decasaccharide AT-10 is the same saccharide used inprevious studies (Rhomberg, A. J., Shriver, Z., Biemann, K. &Sasisekharan, R. (1998) Proc Natl Acad Sci USA 95, 12232-7 and Ernst,S., Rhomberg, A. J., Biemann, K. & Sasisekharan, R. (1998) Proc NatlAcad Sci USA 95, 4182-7). Oligosaccharides were dissolved in deionizedwater at concentrations of 10-35 μM. Heparinase I-III fromFlavobacterium heparinum were purified as described previously. Theexoenzymes α-L-iduronate 2-O sulfatase, α-L-iduronidase,β-D-glucuronidase and N-acetylglucosamine-6-sulfatase were purchasedfrom Oxford Glycosciences. A 40% aqueous solution of sodium nitrite waspurchased from Aldrich Chemical. Disaccharide standards forcompositional analysis were purchased from Sigma-Aldrich (St. Louis,Mo.).

Compositional Analysis. Compositional analysis of oligosaccharides wascompleted by exhaustive digest of a 30 μM sample of AT-10 followed bycapillary electrophoresis (CE) as described previously (Rhomberg, A. J.,Ernst, S., Sasisekharan, R. & Biemann, K. (1998) Proc Natl Acad Sci USA95, 4176-81). Briefly, to 1 nmol of oligosaccharide was added 200 nM ofheparinases I,II, and III in 25 mM sodium acetate, 100 mM NaCl, 5 mMcalcium acetate buffer pH 7.0. The reaction was allowed to proceed at30° C. overnight and then analyzed by CE in reverse polarity with arunning buffer of 50 mM tris/phosphate 10 μM dextran sulfate pH 2.5.

Digests. Heparinase I digestions were designated either short orexhaustive. For short digestion, 50 nM heparinase I was incubated withthe substrate for 10 minutes prior to analysis. Exhaustive digestionswere completed with 200 nM enzyme overnight. Enzyme reactions wereperformed by adding 1 μL of enzyme solution in a buffer containing 10 μMovalbumin, 1 μM dextran sulfate, 5 mM calcium acetate and 10 mMethylenediamine buffer at pH 7.0. to 4 μL of aqueous substrate solution;digestion was allowed to proceed at room temperature as describedpreviously (Venkataraman, G., Shriver, Z., Raman, R. & Sasisekharan, R.(1999) Science 286, 537-42 and Rhomberg, A. J., Ernst, S., Sasisekharan,R. & Biemann, K. (1998) Proc Natl Acad Sci USA 95, 4176-81). Partialnitrous acid cleavage was completed using a modification of publishedprocedures (Turnbull, J. E., Hopwood, J. J. & Gallagher, J. T. (1999)Proc Natl Acad Sci USA 96, 2698-703). Exoenzyme digests were completedeither simultaneously or sequentially. Final enzyme concentrations werein the range of 20-40 milliunits/mL and digestion was carried out at 37°C.

Mass Spectrometry. Mass spectral analyses were carried out on aPerSeptive Biosystems Voyager Elite reflectron time-of-flight instrumentin the linear mode with delayed extraction. Samples from digests wereprepared by removing 0.5 μL of the reaction mixture and adding it to 4.5μL of matrix solution (12 mg/mL caffeic acid in 30% acetonitrile) thatcontained a 2-fold molar excess of the basic peptide (RG)₁₉R (calculatedmass of the (M+H)⁺ ion=4226.8). Addition of the basic peptide tospecifically chelate HLGAG oligosaccharides and mass spectral collectionparameters allow for direct sample analysis without need for samplerepurification (Rhomberg, A. J., Ernst, S., Sasisekharan, R. & Biemann,K. (1998) Proc Natl Acad Sci USA 95, 4176-81). Samples were spotted onthe target and mass spectra were collected using parameters outlinedpreviously (Rhomberg, A. J., Ernst, S., Sasisekharan, R. & Biemann, K.(1998) Proc Natl Acad Sci USA 95, 4176-81). Observed in each massspectrum are the (M+H)⁺ ions of the basic peptide and the (M+H)⁺ ion ofa 1:1 peptide:saccharide complex, and the mass of the saccharide isdetermined by subtracting the measured m/z value of the (M+H)⁺ ion ofthe peptide from that of the 1:1 complex (Juhasz, P. & Biemann, K.(1995) Carbohydr Res 270, 131-47). All spectra on a plate werecalibrated externally using a standard of (RG)₁₉ R and its complex witha nitrous acid-derived hexasaccharide of the sequenceI_(2S)H_(NS,6S)I_(2S)H_(NS;6S)I_(2S)Man_(6S) (calculated mass of 1655.4)under identical instrumental parameters. This methodology requiressufficient sulfation of the saccharide to ensure efficient complexation.As such, small, undersulfated saccharides (i.e., mono- anddisaccharides) are not observed with this methodology (Juhasz, P. &Biemann, K. (1995) Carbohydr Res 270, 131-47).

Integral Glycan Sequencing. Integral glycan sequencing (IGS) usingelectrophoretic separation was carried out as described (Turnbull; J.E., Hopwood, J. J. & Gallagher, J. T. (1999) Proc Natl Acad Sci USA 96,2698-703). Partial nitrous acid cleavage conditions were modified byusing 25 mM HCl and 2.5 mM sodium nitrite and stop time points of 5, 10,20, 30, 120, and 240 minutes.

¹H NMR Spectroscopy. ¹H NMR spectroscopy was performed using theconditions described previously (Nadkarni, V. D., Toida, T., Van Gorp,C. L., Schubert, R. L., Weiler, J. M., Hansen, K. P., Caldwell, E. E. &Linhardt, R. J. (1996) Carbohydr Res 290, 87-96). AT-10 was subjected toion-exchange chromatography to remove paramagnetic impurities. A column(1 cm×10 cm) of AG 50W-X8 (Bio-Rad Japan, Tokyo) was converted intosodium form by treatment with 5 mL of 0.1 M NaOH and washed with waterfor 12 hr before use. The sample for NMR experiments was applied to thecolumn, eluted with 20 mL of water and freeze-dried. The sample (˜1 mg)was then freeze-dried three times from 99.8% D₂O (Merck, Germany) anddissolved in 0.5 mL of 100% D₂O (Aldrich Japan, Tokyo) for NMRspectroscopy in a 5 mm tube. 1D ¹H NMR spectroscopy of AT-10 wasperformed on a JEOL GSX 500A spectrometer equipped with a 5-mm fieldgradient tunable probe at 298K.

Results

Introduction to Sequencing Methodology

Recently a matrix-assisted laser desorption ionization mass spectrometry(MALDI-MS) technique enabling the determination of the mass of HLGAGcomplex oligosaccharides (from di to decasaccharides) to an accuracy ofbetter than ±1 Da was developed (Juhasz, P. & Biemann, K. (1995)Carbohydr Res 270, 131-47 and Juhasz, P. & Biemann, K. (1994) Proc NatlAcad Sci USA 91, 4333-7). Because of the accuracy of the resultingmolecular mass measurement of the individual HLGAGs, a unique assignmentof both the length of a fragment and the number and kind of substituentsis, possible, especially if the oligosaccharide is a tetradecasaccharideor smaller (Venkataraman, G., Shriver, Z., Raman, R. & Sasisekharan, R.(1999) Science 286, 537-42). In addition, MALDI-MS can detectoligosaccharide fragments generated upon enzymatic or chemicaldegradation of anoligosaccharide (Rhomberg, A. J., Shriver, Z., Biemann,K. & Sasiseklaran, R. (1998) Proc Natl Acad Sci USA 95, 12232-7; Ernst,S., Rhomberg, A. J., Biemann, K. & Sasisekharan, R. (1998) Proc NatlAcad Sci USA 95, 4182-7; and Rhomberg, A. J., Ernst, S., Sasisekharan,R. & Biemann, K. (1998) Proc Natl Acad Sci USA 95, 4176-81). Finally,the sensitivity of MALDI-MS is such that as little as 100 femtomoles ofmaterial can be readily detected.

In addition to the MALDI-MS experimental technique, a property-encodednomenclature (PEN) for representing the 32 disaccharide units using ahexadecimal coding system was developed (Venkataraman, G., Shriver, Z.,Raman, R. & Sasisekharan, R. (1999) Science 286, 537-42). Thedevelopment of PEN is necessary to handle the large information content(properties) of HLGAGs. Each of the hexadecimal numbers is derived basedon an internal logic that manifests itself in terms of the distributionof sulfates on a particular building block unit and is not randomlyassigned simply to identify each disaccharide unit. This system isimportant for HLGAGs in that it enables the rapid manipulation ofsequences using simple mathematical or binary operations, henceproviding a handle on the large information content of complexpolysaccharides. In addition, the inherent structural diversity inHLGAGs necessarily arises from the property differences (location ofcharged sulfate and acetate groups) thereby making PEN a naturalassignment scheme for HLGAGs. This is in direct contrast to thealphabetic codes used to represent the nucleotides of DNA and aminoacids or proteins that serve as mere identifiers and do not code for anyinformation (properties) and do not capture the chemical heterogeneityof these biopolymers.

The hexadecimal coding system comprises the alphanumerals 0-9 and A-F.Since the disaccharide unit has 4 positions viz. 2-O, N-, 3-O and 6-Othat can be modified, it is straightforward to assign each of the fourbinary digit positions of the hexadecimal code, to one of these chemicalpositions. In addition, since there are only two modifications possibleat each position, (2-O, 3-O and 6-O can either be sulfated or free, andthe N-position can be sulfated or acetylated, *), the use of a binarysystem captures these modifications as simple on or off states. Forexample, if, within a given disaccharide unit, the 2-O position issulfated, then it is assigned the binary value of 1. Conversely, if the2-O position on a given disaccharide is unsulfated, then it is assignedthe binary value of 0.* There are some rare HLGAG sequences with unsubstituted N-position,which can be accounted for in the PEN system by adding extra bits.However, in our studies, initial experiments including compositionalanalysis (see below) did not show the presence of free amine containingdisaccharides.

To identify a disaccharide with an alphanumeric character, the 4 binarypositions have been assigned in the following manner: the 2-O positionwas assigned the leftmost binary position, followed by the 6-O, 3-O andN-position in that order. In each case, as outlined above, the binarycode 1 was used to represent sulfated positions and 0 was used torepresent unsulfated positions in the case of 2-O, 6-O and 3-O, andacetylation in the case of N position.

To code for the isomeric state of the uronic acid (i.e., iduronic vs.glucuronic acid), we designated disaccharide units as +/−. In this way,it is possible to assign the positive hexadecimal codes to iduronic acidcontaining units and the negative hexadecimal codes to glucuronic acidcontaining units. Thus, disaccharide units with the same hexidecimalcode but opposite signs possess the same sulfation pattern, differingonly in the isomeric state of the uronic acid. Table 1 outlines the useof PEN for the disaccharide units present in this study. TABLE 1Derivation of PEN for Disaccharide Units Used in this Study I/G 2X 6X 3XNX HEX DISACC MASS 0 0 1 0 0 4 I-H_(NAc, 6S) 459.4 0 0 1 0 1 5I-H_(NS, 6S) 497.4 0 1 1 0 1 D I_(2S)-H_(NS, 6S) 577.5 1 0 1 0 1 −5G-H_(NS, 6S) 497.4 1 0 1 1 1 −7 G-H_(NS, 3S, 6S) 577.4

The hexadecimal code derived for the disaccharide units occurring inAT-10 are shown in Table 1. Column 1 is the binary position that codesfor the isomeric state of the uronic acid. Columns 2 through 5 code forthe modifications at the 2-O, 6-O, 3-O and N-positions of thedisaccharide unit. Column 6 shows the hexadecimal codes represented bythe binary digits in columns 2 through 5. Column 7 shows thedisaccharide unit represented by the code in column 6. Column 8 showsthe calculated theoretical masses of the disaccharide unit presentinternally in a sequence. For chemical or enzymatic modifications tothese disaccharides, the following nomenclature is used: uronic acidwith a Δ⁴⁻⁵ unsaturated linkage (ΔU)=±; reducing end disaccharide unitwith a mass tag=^(t); disaccharide unit with a five-memberedanhydromannose ring=′;

Thus, the strategy for the sequence assignment of HLGAG oligosaccharidesby PEN-MALDI essentially involves the following steps. First, MALDI-MSof the intact oligosaccharide is used to assign the length as well asthe total number of sulfates and acetates present in theoligosaccharide. Compositional analysis is then used to determine thenumber and type of disaccharide building blocks. With this information,a master list is constructed of all possible sequences that containthose disaccharide units. In this manner, no sequences are excluded fromthe analysis, no matter how unusual a given sequence may be. The mass ofoligosaccharide fragments generated from enzymatic digestion or chemicaldegradation are applied as experimental constraints and sequences thatdo not satisfy these constraints are eliminated. In an iterative manner,moving from experimental constraints to the ever-decreasing master listof possible sequences, one can rapidly arrive at a unique sequencesolution using a minimum of material. Importantly, multiple pathways,using separate experimental constraints, can be used to converge on asequence, ensuring assignment accuracy.

Analysis of AT-10

AT-10 and all oligosaccharides derived from it either upon enzymatic orchemical treatment are detected with MALDI-MS as non-covalent complexeswith the basic peptide (RG)₁₉R (Rhomberg, A. J., Ernst, S.,Sasisekharan, R. & Biemann, K. (1998) Proc Natl Acad Sci USA 95, 4176-81and Juhasz, P. & Biemann, K. (1995) Carbohydr Res 270, 131-47). Usingthis methodology, two species are observed, a (M+H)⁺ ion of (RG)₁₉R anda (M+H)⁺ ion for the peptide:saccharide complex. The molecular mass ofan oligosaccharide is obtained by subtracting the (M+H)⁺ value of thepeptide from the (M+H)⁺ value of the 1:1 saccharide:peptide complex.Table 2 lists all fragments observed in this study, their calculated andexperimentally derived mass values, and the deduced. structure of thefragments after sequence assignment of AT-10. FIG. 1. shows that themajor component of AT-10 has a m/z value of 6999.3. When the m/z valueof the protonated peptide is subtracted, the experimental value for themass of this oligosaccharide is found to be 2770.2 which can uniquely beassigned to a decasaccharide with 13 sulfates and 1 acetate group. Themass spectrum of AT-10 indicates the presence of another species(hereafter referred to as the contaminant) of mass 2690.1 (aftersubtraction of the peptide contribution), corresponding to anoligosaccharide with 12 sulfates and 1 acetate group. TABLE 2 m/z valuesfor the peaks in the mass spectra and their deduced structures Complex(M + H)⁺ Saccharide (Observed) Deduced Structure Mass (Calculated)6999.3 2770.2ΔU_(2S)H_(NS),_(6S)I_(2S)H_(NS,6S)I_(2S)H_(NS,6S)IH_(NAc,6S)GH_(NS,3S,6S)(FIG. 1) 2769.3 6919.2 2690.1ΔU_(2S)H_(NS),_(6S)I/GH_(NS,6S)I_(2S)H_(NS,6S)IH_(NAc,6S)GH_(NS,3S,6S)(FIG. 1) 2689.2 6899.6 2673.0ΔU_(2S)H_(NS),_(6S)I_(2S)H_(NS,6S)I_(2S)H_(NS,6S)IH_(NAc,6S)GMan_(3S,6S)(FIG. 4) 2672.2 6435.8 2209.2I_(2S)H_(NS),_(6S)I_(2S)H_(NS,6S)IH_(NAc,6S)GH_(NS,3S,6S) (FIG. 4)2209.8 6419.7 2192.2ΔU_(2S)H_(NS),_(6S)I_(2S)H_(NS,6S)IH_(NAc,6S)GH_(NS,3S,6S) (FIG. 2a)2191.8 6339.8 2113.2I_(2S)H_(NS),_(6S)I_(2S)H_(NS,6S)IH_(NAc,6S)GMan_(3S,6S) (FIG. 4) 2112.75899.9 1671.4 ΔU_(2S)H_(NS),_(6S)IH_(NAc,6S)GH_(NS,3S,6S)d* (masstagged, FIG. 2c) 1670.4 5859.8 1633.2I_(2S)H_(NS),_(6S)IH_(NAc,6S)GH_(NS,3S,6S) (FIG. 4) 1632.3 5842.1,5842.2, 5843.6 1614.6, 1614.4, 1615.1ΔU_(2S)H_(NS),_(6S)IH_(NAc,6S)GH_(NS,3S,6S) (FIG. 2a, b, c) 1614.35383.1, 5382.5 1155.6, 1154.0 ΔU_(2S)H_(NS),_(6S)I_(2S)H_(NS,6S) (FIG.2a, c) 1154.9 5301.7 1073.9 ΔU_(2S)H_(NS),_(6S)I/GH_(NS,6S) (FIG. 2b)1074.9 5284.5 1057.9 ΔU_(2S)H_(NS),_(6S)I_(2S)Man_(6S) (FIG. 4) 1057.85241.5 1013.8 IH_(NAc,6S)GMan_(3S,6S)d (mass tagged, FIG. 3) 1013.95186.5  958.8 IH_(NAc,6S)GMan_(3S,6S) (FIG. 3) 957.8 5007.8  780.8H_(NAc,6S)GMan_(3S,6S) (FIG. 3) 780.7 4805.2, 4805.3, 4805.2 577.7,577.5 576.7 ΔU_(2S)H_(NS),_(6S) (FIG. 2a, b, c) 577.5*d - represents the semicarbazide mass tag (Δ = 56.1 daltons)

Shown in column 1 is the m/z value of the protonated 1:1 complex of thesaccharide and the basic peptide (RG)₁₉R. Column 2 shows the observedmass of the saccharide obtained by subtracting the value of theprotonated peptide observed in the spectrum from the protonated 1:1complex. The deduced chemical structures of the 10 saccharides for thecorresponding peaks in the mass spectra are shown in column 3.

Shown in column 4 are the theoretical masses calculated for the deducedstructures. Note that the observed mass (col. 2) is always within ±1dalton of the calculated mass (col. 4).

Compositional analysis using CE indicates the presence of fourdisaccharide. building blocks, corresponding to ΔU_(2S)-H_(NS,6S) (±D),ΔU-H_(NAc,6S) (±4), ΔU-H_(NS,6S) (±5), and ΔU-H_(NS,3S,6S) (±7), in therelative ratio of 2.90:1.00:1.05:0.15 respectively. Thus, compositionalanalysis of this sample confirmed that there are two species, one major(˜85%) and one minor (˜15%). AT-10 must be a decasaccharide made of thebuilding blocks ΔU_(2S)-H_(NS,6S) (±D), ΔU-H_(NAc,6S) (±4), andΔU-H_(NS,3S,6S) (±7) in a ratio of 3:1:1. Together, the CE and MALDI-MSdata was used to construct a master list of possible sequences forAT-10. We find that 320 sequences can account for both the CE and MSdata (Table 2). These 320 sequences constitute the master list fromwhich sequences were eliminated based on experimental constraints untilconvergence at a single solution.

In addition, the compositional analysis confirmed that there is acontaminant present that was structurally similar to AT-10, except forthe presence of ΔU-H_(NS,6S) (±5). From the CE data, the composition ofthe contaminant was determined to be ΔU_(2S)-H_(NS,6S) (±D),ΔU-H_(NAc,6S) (±4), ΔU-H_(NS,3S,6S) (±7), and ΔU-H_(NS,6S) (±5) in therelative ratio of 2:1:1:1 from successive subtraction.

Having constructed the master list of sequence possibilities, acombination of PEN-MALDI, IGS (Turnbull, J. E., Hopwood, J. J. &Gallagher, J. T. (1999) Proc Natl Acad Sci USA 96, 2698-703), and NMRanalysis was used to sequence AT-10 and then to analyze the sequence ofthe contaminant.

MALDI-MS Sequencing of AT-10

From the list of 320 possible sequences generated from the compositiondata, we have used a series of experimental constraints, including theuse of heparinase I and nitrous acid, respectively, to assign thesequence of AT-10.

Short (incomplete) digestion of AT-10 with heparinase I results in fivefragments of molecular mass 577.7, 1073.9, 1155.6, 1614.6 and 2192.2(FIG. 2 a). The fragment with mass 577.7 corresponds to ±D. The 1155.6fragment corresponds to a hexasulfated tetrasaccharide which has to haveone: of the following structures: ±DD, ±D-D, ±D7, ±D−7, ±7D, or ±7−D.The fragment with 1614.6 corresponds to a heptasulfated monoacetylatedhexasaccharide and the fragment with 2192.2 corresponds to adecasulfated monoacetylated octasaccharide. The last peak, at 1073.9 wasassigned unambiguously to the contaminant (see below for analysis). Whenthe list of 320 sequences were searched for these fragments formed bysimulated heparinase I digestion, it reduced the list to 52 sequences(Table 2).

The rate of substrate cleavage by heparinase I is size dependent(Linhardt, R. J., Turnbull, J. E., Wang, H. M., Loganathan, D. &Gallagher, J. T. (1990) Biochemistry 29, 2611-7). To identify all 2-Osulfated iduronate-containing linkages in AT-10, it was treated withheparinase I under conditions that resulted in complete cleavage of allsusceptible linkages. Under these conditions, the hexasaccharide andtetrasaccharide, from the contaminant, remained intact (FIG. 2 b).However, the hexasulfated tetrasaccharide (mass of 1155.6 from FIG. 2 a)was cleaved. Thus, this saccharide has the sequence ±DD. Of the 52possible sequence. assignments for AT-10, only 28 can satisfy theheparinase I exhaustive digest data.

Next, AT-10 was treated with semicarbazide to yield a semicarbazone atthe anomeric position. In this fashion a mass tag (Δ=56.1) wasintroduced to differentiate fragments derived from the reducing end asopposed to the non-reducing end. Treatment of tagged AT-10 withheparinase I yielded five fragments (FIG. 2 c). From a comparison ofheparinase I-treated underivatized AT-10 (FIG. 2 a), the heptasulfatedmonoacetylated hexasccharide appears tagged and thus must be derivedfrom the reducing end of AT-10. Application of this constraint to the 28remaining sequences eliminates all but 12 of them (Table 3). TABLE 3Convergence of the AT-10 sequence

The stepwise strategy used to sequence the decasaccharide sample isshown in Table 3. Application of experimental constraints to eliminatethe sequences from the master list of 320 sequences was used to convergeto the final sequence. Shown in the boxes on the left is the number ofsequences that satisfied the experimental constraints. The boxes on theright show the sequences that satisfy the experimental constraints alongwith the possible fragments formed for the masses shown in parenthesison the top of the table.* To obtain all the possible decasaccharides with the composition of3±Ds, a ±4 and a ±7 we need to arrange the above disaccharide units inall the possible ways, to form a decasaccharide. Also each disaccharideunit can be a + or a − corresponding to iduronate or glucuronate. Thenumber of possible sequences=⁵C₃ (arrange 3Ds in 5 positions)*²C₁(arrange the 4 in the two remaining positions)*2⁴ (to account for the +or − at all the positions except the non-reducing end, since thesaccharide is heparinase derived)=10*2*16=320.

Inspection of the 12 remaining sequences in Table 3 indicates that theprimary difference is in the identity of the reducing-endhexasaccharide. Therefore, through nitrous acid degradation of AT-10 andjudicious use of exoenzymes, the sequence of the reducing endtetrasaccharide was determined. First, tagged AT-10 was exhaustivelytreated with nitrous acid and two species were readily detectable (FIG.3). The first, with a mass of 1013.8, corresponds to a taggedanhydromannose tetrasaccharide with four sulfates and one acetate. Theother with a mass of 958.8 corresponds to the same tetrasaccharide thatis untagged. Both could be assigned to one of the following sequences:±47, ±4−7, ±4−D. Thus, based on this information, half of the possiblesequences could be eliminated, leaving only 6 possible sequencesolutions for AT-10 (Table 3).

To assign uniquely the isomeric state of the two disaccharide units atthe reducing end of AT-10, the following experimental constrains wereused: the exhaustive nitrous acid digest was incubated with the exolyticenzyme α-iduronidase that specifically clips the iduronic acid at thenon-reducing end. A shift in the spectrum by 178.0 confirmed the uronicacid as 4 and its isomeric state as +, i.e., IH_(NAc,6S) or +4. Only 3sequences could give the observed fragments, viz., ±7DD4−D, ±DDD4−7,±DDD47.

To distinguish among these last three alternatives and to identify thereducing end disaccharide, the iduronidase-treated product was firsttreated with 6-O sulfatase and N-deacetylase to remove the hexosamine,leaving only the reducing end disaccharide. Treatment of this samplewith β-glucuronidase resulted in degradation to monosaccharides. Thisidentified the reducing end disaccharide as −7 (G-H_(NS,3S,6S)). Thusthe deduced sequence of the AT-III fractionated decasaccharide is±DDD4−7(ΔU_(2S)H_(NS,6S)I_(2S)H_(NS,6S)I_(2S)H_(NS,6S)IH_(NAc,6S)GH_(NS,3S,6S)).Of note is the fact that this sequence does not agree with the sequenceassignment for a decasaccharide produced in an identical manner (Toida,T., Hileman, R. E., Smith, A. E., Vlahova, P. I. & Linhardt, R. J.(1996) J. Biol Chem 271, 32040-7):. Therefore, we sought to confirm oursequencing assignment sing other analytical methodologies.

IGS Sequencing of AT-10.

AT-10 was also sequenced using the recently established techniqueIntegral Glycan Sequencing (IGS), which employs an electrophoreticseparation of saccharides tagged at the reducing end with a fluorophore.Partial nitrous acid cleavage and exoenzyme digestion of the saccharideproduces a ladder from which the sequence can be determined. In accordwith the PEN-MALDI data, electrophoretic analysis of thefluorophore-tagged sample produced a single major decasaccharide, but inaddition, the smaller contaminant was also evident. The products ofpartial nitrous acid cleavage were deca-, octa-, hexa-, andtetrasaccharides, with no disaccharide products observed. This resultdefines the positions of all of the NS and NAc moieties, with just oneN-acetylated disaccharide in the position proximate to the reducing end.Gel shifts due to treatment of these products with differentcombinations of exoenzymes demonstrated iduronate residues in 3positions, 2 of which were 2-O-sulfated, and the presence in threepositions of 6-O-sulfated glucosamine residues. The non-reducing end wasclearly 2-O-sulfated but confirmation of the presence a 6-O-sulfate onthe non-reducing end glucosamine residue, and details of the sulfationpattern on the reducing end monosaccharide were not obtained in thisanalysis. This data defines the structure of the AT-10 asΔU_(2S)H_(NS,±6S)I_(2S)H_(NS,6S)I_(2S)H_(NS,6S)IH_(NAc,6S)GH_(NAc/Ns,±3S,±6S)This data, derived from an independent sequencing approach, is entirelyconsistent with the PEN-MALDI analysis.

Sequence Analysis of AT-10 Contaminant

The mass spectral data can also be used in conjunction with the: CEcompositional analysis to arrive at a proposed sequence for the 12sulfated, 1 acetylated contaminant of AT-10. As stated above, heparinaseI digest (FIG. 2 a) yielded a peak at m/z of 1073.9 that corresponds toa pentasulfated tetrasaccharide (±D±5, ±7±5, or ±5±7), that isassignable only to the contaminant. This tetrasaccharide fragment wasnot derived from the reducing end of the contaminant since under noconditions was a labeled saccharide containing ±5 found. In addition, aheparinase I digest of tagged decasaccharide places 4−7 at the reducingend for both the contaminant as well as for AT-10. To place the positionof the D5 or D−5 tetrasaccharide observed in the heparinase I digest thedecasaccharide was treated with iduronate 2-O prior to heparinase Itreatment. Under these conditions, the pentasulfated tetrasaccharidereduced in mass by 80 Da (from mass of 1073.9 to 993.9, resulting fromthe loss of sulfate). Therefore, this tetrasaccharide must be derivedfrom the non-reducing end of the contaminant. Together, this informationsuggests that the sequence of the contaminant is ±D5D4−7 or ±D−5D4−7.

The assignment for AT-10 and the contaminant was confirmed when thedecasaccharide was incompletely degraded with nitrous acid (FIG. 4).AT-10 with an anhydromannose at the reducing end (mass of 2673.0) isclearly observed as are fragments resulting from nitrous acid scission(masses of 2209.2, 2113.2 and 1633.2) of AT-10. In addition, a specieswith mass 1057.9 can only be obtained fromΔU_(2S)H_(NS,6S)I_(2S)Man_(6S), providing a unique mass signature of thenon-reducing end of AT-10. Importantly, all of the species could beassigned to either AT-10 or the contaminant.

Interpretation of NMR Spectrum of AT-10

The NMR spectrum of AT-10 is shown in FIG. 6. Consistent with ouranalysis, the small signals of αH-1, αH-2 and αH-3 of H_(NS,3S,6S) (at5.45, 3.45 and 4.50 ppm, respectively) allow us to assign the reducingend monosaccharide unit as H_(NS,3S,6S). In this case, the anomericproton of the reducing end H_(NS3S6S) residue must be split into α andβ-configurations. The α-configuration of the anomeric proton of theH_(NS) residue is dominant (˜95%) in protic solvents, such as deuteriumoxide, based on the anomeric effect. Furthermore, the presence of twoI_(2S) moieties could be detected. Interestingly, the anomeric signalsof the two I_(2S), which usually resonate around 5.20 ppm, were shifted.This most probably results from a change in the conformation of theinternal I_(2S) moiety from ¹C₄ to ²S₀. Also, it could be confirmed thatthe oligosaccharide contains three H_(NS,6S)and one unsulfated G residuebased on the integration values of H-2 protons of H_(NS,6S) and Gresidues observed at 3.28 and 3.38 ppm, respectively. The presence ofone N-acetyl methyl signal of H_(NAc,6S) residue at 2.1 ppm clearlydemonstrates that the oligosaccharide contains one H_(NAc,6S) residue.The presence of signals corresponding to the H-6 protons of 6-O-sulfatedH_(NY) residues (Y=Ac or S) at around 4.3 and 3.9 ppm, confirms that allH_(NY) residues of the oligosaccharide are O-sulfated at C-6. Together,this data allows the sequence assignment of the major species in theAT-10 sample asΔU_(2S)H_(NS,6S)I_(2S)H_(NS,6S)I_(2S)H_(NS,6S)IH_(NAc,6S)GH_(NS,3S,6S).

SUMMARY

It has been shown in this Example that several rigorous analyticaltechniques can be used to converge on the structure of a complex HLGAGoligosaccharide. Furthermore, it was demonstrated that the sequenceassignment using the two sequencing procedures, viz., IGS and PEN-MALDIis the same. This is most apparent in the partial and exhaustive nitrousacid treatment of the decasaccharide (FIG. 3). Importantly, from ouranalysis it now becomes possible to assign the following sequences tothe deca, octa, hexa, and tetrasaccharides observed upon gelelectrophoresis and MALDI-MS, viz. DDD4−7, DD4−7, D4−7, and a nitrousacid resistant tetrasaccharide 4−7. In the next Example, the functionalconsequences of a partially intact AT-III binding site was explored andthe enzymatic action of the heparinases towards the AT-III binding site.

In addition, it was demonstrated that the PEN-MALDI approach issufficiently sensitive and discriminating to allow us to determinesequence information for a oligosaccharide mixture. It is essential topoint out the fact that convergence to a single solution for AT-10 usingPEN-MALDI is possible using multiple orthogonal experimental constraints(Venkataraman, G., Shriver, Z., Raman, R. .& Sasisekharan, R. (1999)Science 286, 537-42), thus minimizing reliance on a single experimentalconstraint, e.g., nitrous acid cleavage. Finally, the example shownillustrates the value of PEN-MALDI for obtaining definitive sequenceinformation for biologically and pharmacologically relevantoligosaccharides.

Example 2 Cleavage of the Antithrombin III Binding Site in Heparin byHeparinases and Its Implication in the Generation of Low MolecularWeight Heparin

Introduction:

Heparin has been used as a clinical anticoagulant for over 50 years,making it one of the most effective pharmacological agents known. Muchof heparin's activity can be traced to its ability to bind antithrombinIII (AT-III). Low molecular weight heparin (LMWH), derived from heparinby its controlled breakdown, maintains much of the antithromboticactivity of heparin without many of the serious side effects. Theclinical significance of LMWH has highlighted the need to understand anddevelop chemical or enzymatic means to generate it. The primaryenzymatic tools used for the production of low molecular weight heparinare the heparinases from Flavobacterium heparinum, specificallyheparinases I and II. Using pentasaccharide and hexasaccharide modelcompounds, we show that heparinase I and II, but not heparinase III,cleave the AT-III binding site. Furthermore, we show herein thatglucosamine 3-O sulfation at the reducing end of a glycosidic linkageimparts resistance to heparinase I, II and III cleavage. Finally, weexamine the biological and pharmacological consequences of a heparinoligosaccharide. We show that such an oligosaccharide lacks some of thefunctional attributes of HLGAG containing an intact AT-IlI site.

Methods:

Materials. Penta 1 and 2 was a generous gift of Dr. Robert Rosenberg,Department of Biology, MIT. Hexa 1 was generated using heparinase Idigestion of heparin (Ernst, S., Langer, R., Cooney, C. L. &Sasisekharan, R. (1995) Crit Rev Biochem Mol Biol 30, 387-444). Heparinwas purchased from Celsus Laboratories (Cinncinati, Ohio) and molarconcentrations of stocks were calculated based on an average molecularweight of 13,000 Da. Enoxaparin was purchased from AvantisPharmaceuticals, (Chicago, Ill.).

Digests. Heparinase I digests were completed as described.(Venkataraman,G., Shriver, Z., Raman, R. & Sasisekharan, R. (1999) Science 286, 537-42and Rhomberg, A. J., Ernst, S., Sasisekharan, R. & Biemann, K. (1998)Proc Natl Acad Sci USA 95, 4176-81). Heparinase II or III reactions werecompleted in essentially the same way at room temperature in 10 μMovalbumin, 1 μM dextran sulfate, and 10 mM ethylenediamine, pH 7.0.Short digestions were completed with 50 nM enzyme for 10 minutes whileexhaustive digests were completed with 200 nM enzyme overnight. Massspectra were collected using parameters as outlined above (see alsoShriver, Z., Raman, R., Venkataraman, G., Drummond, K., Turnbull, J.,Toida, T., Linhardt, R., Biemann, K. & Sasisekharan, R. (2000) Proc NatlAcad Sci USA, 2000 Sept. 12; 97(19):10359-64) and calibrated externallyby using signals for protonated (RG)₁₉R and its complex with a nitrousacid-derived hexasaccharide of the sequence I_(2S)H_(NS,6S)I_(2S)H_(NS,6S) I_(2S)Man_(6S)

Equilibrium Fluorescence Titration Experiments. Titrations of humanAT-III with either AT-10 decasaccharide or heparin were completed at 25°C. using a Fluorolog 2 machine (Spex Instruments) (Meagher, J. L.,Beechem, J. M., Olson, S. T. & Gettins, P. G. (1998) J Biol Chem 273,23283-9 and Desai, U. R., Petitou, M., Bjork, I. & Olson, S. T. (1998) JBiol Chem 273, 7478-87). Measurements were completed in 20 mM sodiumphosphate, containing 0.1 mM EDTA and 0.1% PEG 8000, adjusted to eitherpH 7.4 or 6.0. With the pH 7.4 buffer, sodium chloride was added to afinal concentration of 100 mM.

Fluorescence emission spectra were collected from 300-400 nm with a 280nm excitation wavelength and a 5 s integration time. Briefly, thetitration experiments were conducted as follows—aliquots of eitherdecasaccharide or heparin was added to a 1 μM solution of AT-III, thesolution was allowed to come to equilibrium for one minute, and anemission spectrum collected. Addition of sequential saccharide aliquotsand fluorescence signal was adjusted to account for protein dilution.

Biological Measurements of Decasaccharide Activity. In vitroanticoagulant activity was determined as described previously(Hoppensteadt, D. A., Jeske, W. P., Walenga, J. M., Fu, K., Yang, L. H.,Ing, T. S., Herbert, J. M. & Fareed, J. (1999) Thromb Res 96, 115-24 andDietrich, C. P., Paiva, J. F., Castro, R. A., Chavante, S. F., Jeske,W., Fareed, J., Gorin, P. A., Mendes, A. & Nader, H. B. (1999) BiochimBiophys Acta 1428, 273-83), according to the United StatesPharmacopoeia. Thrombin (FIIa) and factor Xa (FXa) generation inhibitionassays were completed essentially as described. Briefly, either AT-10decasaccharide, Enoxaparin LMWH or the synthetic AT-III bindingpentasaccharide (Penta 1) used in this study was dissolved in sterilesaline at the designated concentrations. To this sample was added anequal volume of fibrinogen deficient plasma diluted 1:8 in 100 mMTris-HCl (pH 8.5). In a separate sample, the same concentration ofheparin oligosaccharide and actin was added in a 1:1 ratio to eitherSpectrozyme TH or FXa. In this manner, the intrinsic IIa and Xageneration was measured. In addition, to account for inhibition ofthrombin and extrinisic generation of FXa, thromboplastin C was diluted1:6 with either -Spectrozyme TH or FXa. For all samples, the opticaldensity was measured at 405 nm and results are expressed as a %inhibition compared to a unsupplemented saline control. For theseassays, thrombin reagent (Fibrindex) was obtained from Ortho DiagnosticSystems, Inc. (Raritan, N.J.), and factor Xa was obtained from EnzymeResearch (South Bend, Ind.). Spectrozyme TH and FXa were obtained fromAmerican Diagnostica (Greenwich, Conn.).

Whole blood data were also used to determine the anticoagulant activityof AT-10. The assays, activated partial thromboplastin time (APTT) andprothrombin time (PT) was conducted in a manner similar to what has beenpreviously reported (Dietrich, C. P., Paiva, J. F., Castro, R. A.,Chavante, S. F., Jeske, W., Fareed, J., Gorin, P. A., Mendes, A. &Nader, H. B. (1999) Biochim Biophys Acta 1428, 273-83). APTT reagent wasobtained from Organon Teknika (Durham, N.C.) and Hep Test Reagent wasobtained from Haemachem (St. Louis, Mo.).

Results:

Enzymatic Action of the Heparinases Towards the AT-III Binding Site:Previously, we investigated the substrate specificity of heparinases Iand II towards AT-10 (Rhomberg, A. J., Shriver, Z., Biemann, K. &Sasisekharan, R. (1998) Proc Natl Acad Sci USA 95, 12232-7 and Ernst,S., Rhomberg, A. J., Biemann, K. & Sasisekharan, R. (1998) Proc NatlAcad Sci USA 95, 4182-7). These studies, however, were carried outassuming a published structure (Toida, T., Hileman, R. E., Smith, A. E.,Vlahova, P. I. & Linhardt, R. J. (1996) J Biol Chem 271, 32040-7). Inlight of the newly determined structure of AT-10 described herein (seealso Shriver, Z., Raman, R., Venkataraman, G., Drummond, K., Tumbull,J., Toida, T., Linhardt, R., Biemann, K. & Sasisekharan, R. (2000) ProcNatl Acad Sci USA, 2000 Sep. 12; 97(19):10359-64), we reexamined theenzymatic action of heparinases I, II, and III towards oligosaccharidescontaining a 3-O sulfate that is important for high affinity AT-IIIbinding. For these studies, we used three oligosaccharides, twopentasaccharides (Penta 1 and Penta 2, FIG. 5) and a hexasaccharide(Hexa 1, FIG. 5). Of note is the fact that the pentasaccharides aresynthetically derived whereas Hexa 1 is derived from treatment ofheparin by heparinase I. As a result, unlike Penta 1 or Penta 2, Hexa 1contains a Δ^(4,5) uronic acid at the non-reducing end. Furthermore,Penta 1 and Penta 2 differ from one another only by the presence(Penta 1) or absence (Penta 2) of a 3-O sulfate on the internalglucosamine residue (FIG. 5). The strategy employed herein essentiallyinvolves treatment of each of the saccharides with heparinase I, II, orIII, respectively, under exhaustive digestion conditions, followed bythe identification of the resulting products by mass spectrometry. Thecalculated mass of the-saccharide substrates and products, theiridentity, and the observed mass is listed in Table 4. TABLE 4 Chemicalstructures and m/z values of the HLGAG oligosaccharides Complex (M + H)⁺Saccharide (mass) Chemical Structure Mass (Calculated) 5842.0 1615.2ΔU_(2S)H_(NS,6S)IH_(NAc,6S)GH_(NS,3S,6S) 1614.3 5735.1 1508.3H_(NS,6S)GH_(NS,3S,6S)I_(2S)H_(NS,6S,OMe) 1508.2 5655.1 1428.3H_(NS,6S)GH_(NS,6S)I_(2S)H_(NS,6S,OMe) 1428.1 5316.3 1089.5ΔUH_(NS),_(6S)I_(2S)H_(NS,6S,OMe) 1088.9 5266.3, 5264.2 1036.5, 1036.1ΔUH_(Nac),_(6S)GH_(NS,3S,6S) 1036.9 5064.1  837.3 H_(NS,6S)GH_(NS,6S) 836.7 5143.4, 5144.0 916.7, 917.3 H_(NS,6S)GH_(NS,3S,6S)  916.8 4818.0,4818.0, 591.3, 591.3, ΔU_(2S)H_(NS,6S,OMe)  591.5 4818.7, 4818.7 591.9,592.0 4807.2, 4805.9 577.4, 577.8 ΔU_(2S)H_(NS),_(6S)  577.5

Shown in column 1 of Table 4 is the m/z value of the protonated 1:1complex of the saccharide and the basic peptide (RG)₁₉R. Column 2 showsthe observed mass of the saccharide obtained by subtracting the mass ofprotonated basic peptide from the protonated 1:1 complex. The chemicalstructures of the saccharides for the corresponding peaks in the massspectra are shown in column 3. Column 4 shows the theoretical massescalculated for the chemical structures. Note that the observed mass iswithin ±1 Da of the calculated mass.

In the case of Penta 1, only heparinase I and II, but not heparinaseIII, cleave the oligosaccharide into a pentasulfated trisaccharide ofmass 916.7 and a trisulfated disaccharide of mass 591.3 (FIG. 6),indicative of cleavage at the I_(2S)-containing glycosidic linkage(linkage A.2 in FIG. 5). The data presented in FIG. 6 show that 3-Ocontaining linkages are resistant to heparinase I, II or III cleavage.This resistance appears to be length independent. Based on the previousunderstanding of the structure of AT-10 (Toida, T., Hileman, R. E.,Smith, A. E., Vlahova, P. I. & Linhardt, R. J. (1996) J Biol Chem 271,32040-7), it was reported that heparinase II could cleave a 3-O sulfatecontaining saccharide provided that it was of sufficient length(Rhomberg, A. J., Shriver, Z., Biemann, K. & Sasisekharan, R. (1998)Proc Natl Acad Sci USA 95, 12232-7). In light of the newly determinedstructure for the AT-10 decasaccharide (See also Shriver, Z., Raman, R.,Venkataraman, G., Drummond, K., Turnbull, J., Toida, T., Linhardt, R.,Biemann, K. & Sasisekharan, R. (2000) Proc Natl Acad Sci USA, 2000 Sep.12; 97(19):10359-64), as well as the data presented in FIG. 6, theprevious findings must be reinterpreted and it is concluded herein thatheparinase II does not cleave linkages with a reducing-end proximate 3-Osulfated glucosamine. In addition, resistance of this linkage toheparinase I, II and III action is entirely due to the presence of a 3-Osulfate as shown by heparinase I, II, and III treatment of Penta 2 (FIG.7). In this case, all of the heparinases efficiently cleave thesubstrate. As with Penta 1, heparinase I cleaves at theI_(2S)-containing linkage yielding a trisaccharide of mass 837.3 and adisaccharide of mass 591.9 (cleavage at linkage B.2 in FIG. 5).Conversely, both heparinase II and III cleave at the now scissileunsulfated G-containing linkage (linkage B.1 in FIG. 5). Heparinase IIIcleaves only this linkage giving a tetrasaccharide of mass 1428.3 and amonosaccharide (not observed). Heparinase II cleaves at linkage B.2 aswell as B.1, reducing Penta 2 to a monosaccharide and two disaccharides,one of which his observed and has a mass of 592.0 (FIG. 7).

To explore further the substrate specificity of the heparinases towardsthe 3-O sulfated saccharides, we used Hexa 1, a 3-O sulfate-containing,heparinase I-derived hexasaccharide (FIG. 8). Hexa 1 was also chosen asa substrate for this study because it represents a non-reducing endtruncation of AT-10 and contains the same GH_(NS,3S,6S) moiety at thereducing end. We find that hexa 1 is susceptible to heparinase II andIII cleavage but not heparinase I scission. Of note is the fact thatcleavage of Hexa 1 by either heparinase II or III does not occur at theG-containing linkage but rather at the I-containing linkage (cleavage atC.1 but not C.2 in FIG. 5). In the case of heparinase II cleavage, theproducts are a tetrasaccharide of mass 1036.5 and a disaccharide of mass577.4., For heparinase III cleavage, the same products are observed,viz., a tetrasaccharide of mass 1036.1 and a disaccharide of mass 577.8.These results confirm our assessment that linkages with a reducing endproximate 3-O sulfate are protected from heparinase action, includingheparinase II. With both heparinase II and III treatment of Hexa 1, aheparinase-resistant tetrasaccharide, with the sequenceΔUH_(NAC,6S)GH_(NS,3S,6S) is formed. That this tetrasaccharide isresistant to further heparinase cleavage is consistent with previousobservations (Yamada, S., Yoshida, K., Sugiura, M., Sugahara, K., Khoo,K. H., Morris, H. R. & Dell, A. (1993) J Biol Chem 268, 4780-7). Alsoconsistent with the known substrate specificity of heparinase I, Hexa 1,which contains no I_(2S) linkages, is not cleaved by this enzyme.

In light of these studies, it is now apparent that heparinases I and IIcan cleave the AT-III site at the H_(NS,3S,6S)⇓I_(2S)H_(NS,6S) linkage,where the scissile linkage is designated with an arrow (A.2 of Penta 1and B.2 of Penta 2, FIG. 5). Furthermore, with the newly assignedstructure for AT-10, we find that linkages with a 3-O sulfate on thereducing end glucosamine, viz., H_(NS,6S)⇓GH_(NS,3S,6S) are notcleavable by either heparinase II or III (A.1 of Penta 1 and B.1 ofPenta 2, FIG. 5). In addition, we find that this inhibition is entirelydue to the unusual 3-O sulfate modification. Finally, taken togetherwith our previous studies of the enzymatic action of heparinase I, theAT-10 structure reinforces the fact that heparinase I acts in anexolytic, processive manner on heparin/heparan oligosaccharides (Ernst,S., Rhomberg, A. J., Biemann, K. & Sasisekharan, R. (1998) Proc NatlAcad Sci USA 95, 4182-7).

AT-III Bindiug to the AT-10 Decasaccharide: Our sequence analysis ofAT-10 reported herein and in Shriver, Z., Raman, R., Venkataraman, G.,Drummond, K., Turnbull, J., Toida, T., Linhardt, R., Biemann, K. &Sasisekharan, R. (2000) Proc Natl Acad Sci USA, 2000 Sep. 12;97(19):10359-64 revealed that this decasaccharide does not contain anintact AT-III binding pentasaccharide sequence but rather contains onlythe non-reducing end trisaccharide unit. We sought to extend thissequence assignment and provide a functional context to this result bymeasuring the AT-III binding affinity of AT-10. At pH 7.4, I=0.15, AT-10has very little affinity for AT-III (FIG. 9). Conversely, under the sameconditions, porcine intestinal mucosa heparin bound AT-III with anapparent K_(D) of 36 nM. To measure accurately an affinity of AT-10 forAT-III, the titration was completed instead at pH 6.2, conditions thatare known to promote AT-III binding to saccharides. Under theseconditions, AT-10 bound AT-III with an apparent K_(D) of 0.8 μM whilethe K_(D) for full-length heparin decreased to 10 nM. The measured K_(D)for AT-10 is comparable with similar saccharides with a truncatedreducing end, with measured K_(D) values of 0.3-2 μM (Desai, U. R.,Petitou, M., Bjork, I. & Olson, S. T. (1998) J Biol Chem 273, 7478-87).Thus, the results of the titration experiments are consistent with AT-10containing a partially intact AT-III pentasaccharide binding sequence(Desai, U. R., Petitou, M., Bjork, I. & Olson, S. T. (1998) J Biol Chem273, 7478-87). The three saccharide units at the non-reducing end of thepentasaccharide: sequence, viz., H_(NAc,6S)GH_(NS,3S,6S) are primarilyresponsible for binding of the native state of AT-III, while thereducing end disaccharide unit, I_(2S)H_(NS,6S), which is missing inAT-10, is important for binding the active, conformationally-altered ATIII. The measured K_(D) for AT-10 (0.8 μM at pH 6.0, I=0.05) is ˜100times higher than that of full-length heparin, confirming that AT-10does not contain an intact AT-III pentasaccharide sequence. The decreasein AT-III affinity observed for AT-10 cannot be due simply to a sizeissue since, in previous studies, the pentasaccharide alone has beenshown to have an affinity similar to that of full length heparin (Desai,U. R., Petitou, M., Bjork, I. & Olson, S. T. (1998) J Biol Chem 273,7478-87). Having measured the binding interaction between AT-III and thedecasaccharide, we next sought to define the functional consequences ofa HLGAG oligosaccharide that contains only a partial AT-III bindingsite.

Biological Activity of AT-10: As might be expected for a oligosaccharidethat does not contain an intact AT-III site, the biological activity ofAT-10 is less than that of either enoxaparin (used here as an example ofa LMWH) or the pentasaccharide, Penta 1 (FIG. 10). Consistent with theknown mechanism of heparin-mediated inhibition of thrombin activity byAT-III, neither the decasaccharide nor the pentasaccharide havesignificant anti-IIa activity (FIG. 10 a). In the case of thepentasaccharide this lack of activity is entirely due to its size beinginsufficient to act as a template for the formation of a AT-III/IIacomplex. For the decasaccharide, this size constraint is also a probableexplanation (since rigorous biochemical studies have implicatedoligosaccharides with at least 18 monosaccharide units being importantfor efficient complex formation) (Petitou, M., Herault, J. P., Bernat,A., Driguez, P. A., Duchaussoy, P., Lormeau, J. C. & Herbert, J. M.(1999) Nature 398, 417-22), though the lack of an intact AT-III site mayalso contribute to its reduced anti-IIa activity.

These results are confirmed and extended by examining the anti-Xaactivity of the three using factor Xa from serum (FIG. 10 b) or purifiedfactor Xa (FIG. 10 c). As has been shown previously, inhibition ofFactor Xa by AT-III requires binding of the pentasaccharide motif onlyconcomitant with a conformational change in, AT-III. In the anti-Xaassay, both enoxaparin and the pentasaccharide have markedly higheractivity than the decasaccharide. The IC₅₀ values of enoxaparin and thesynthetic pentasaccharide are 66 nM and 39 nM, respectively, while thatof AT-10 is ten-fold higher at 280 nM. These values are consistent withthe lower affinity for AT-III of AT-10 as compared to heparin which wasdetermined in the AT-III fluorescence titration experiments (FIG. 9).That the decasaccharide possesses anti-Xa activity is not surprisingsince the non-reducing trisaccharide unit (present in thedecasaccharide) is primarily responsible for initial binding of heparinto AT-III. The reducing end disaccharide unit, viz., I_(2S)H_(NS,6S)(missing in AT-10) is expected to bind to conformationally alteredantithrombin III, stabilizing it. The Hep Test measurements (FIG. 10 d)yield similar results, viz., enoxaparin and the pentasaccharide havesignificantly higher activity than the AT-10 decasaccharide. Takentogether, the anti-IIa and anti-Xa activities of the decasaccharide ascompared to the pentasaccharide and enoxaparin agree well with theAT-III titration experiments as well as the known pharmacology ofheparin's mechanism of inhibition of the coagulation cascade.

In summary, it is shown in this example that heparinases I and II cleavethe AT-III binding site leaving behind the trisaccharide unit at thereducing end of the oligosaccharide. We also demonstrate that heparinaseIII does not cleave the AT-III site because of the presence of a 3-Osulfate on the internal glucosamine residue. Thus, to use heparinases Ior II for the generation of LMWHs requires extreme caution to ensureretaining intact antithrombin III sites in LMWH fragments. In fact, theresults demonstrated herein show that heparinases I or II may be idealagents for the neutralization of pharmacological doses of heparin.

Example 3 Development of a Compositional Analysis Method and StructuralCharacterization of Heparins

Introduction

To purify UFH and identify its components, UGH was exhaustively digestedand analyzed using capillary electrophoresis and MALDI massspectrometry. Capillary electrophoresis (CE) is a very sensitive methodwith high resolving power for the disaccharide compositional analysis-ofheparins. A compositional analysis method (CAM) using CE for quantifyingthe disaccharide building blocks of UFH and LMWH was developed. Thismethod uses less than a microgram of heparin, is time efficient (each CErun is 25 minutes long), is concentration independent, and is highlyreproducible. One advantage of this method is its effectiveness as aquality control process that may help minimize batch-to-batch variationin the composition of different LMWH

Thus, CE in combination with off line MALDI mass spectrometry has beenused to identify a unique tetrasaccharide in the exhaustive digest ofUFH, and LMWH. This tetrasaccharide, which forms a part of the AT IIIbinding pentasaccharide domain of glycosaminoglycans, is resistant tofurther degradation with Heparinase I, II, or III. It is shown belowthat the utilization of this unique tetrasaccharide in the directmeasurement of heparin's anti factor Xa mediated anticoagulant activity.

Methods

Chemicals and Materials: UFH was purchased from Celsus Laboratories(Cincinnati, Ohio) and molar concentrations of stocks were calculatedbased on an average molecular weight of 13,000 Da. Disaccharidestandards were purchased from Sigma-Aldrich (St. Louis, Mo.). HeparinaseI, and III are recombinant heparinases. Heparinase II is fromFlavobacterium heparinum purified as described previously. (Shriver etal. Journal of Biological Chemistry 1998, 273, 22904-22912.)

Compositional Analysis: UFH was subjected to exhaustive depolymerizationwith an enzyme cocktail made up of Heparinase I, Heparinase II, andHeparinase III. 9 μl of 10 μg/μl concentration of UFH in H₂O wasdigested with 1 μl of Enzyme cocktail consisting of 100 nM each ofheparinase I, II, and III in 25 mM sodium acetate, 100 mM sodiumchloride, 5 mM calcium acetate buffer, pH 7.0 for 12 h at 37° C. The CEsample was prepared by diluting 1 μl of the digest with 9 μl of H₂O. Thesamples were analyzed by CE in reverse polarity with a running buffer of50 mM tris/phosphate, 10 μM dextran sulfate, pH 2.5. 57 nL of eachsample was injected into the CE and run times were 25 minutes. Eachsample was digested in duplicate and the experiment was repeated twicefor each sample, resulting in four readings per sample. All of the 8resulting peaks were collected, and the purity of the collected sampleswas checked by re-injecting into CE, and their mass was measured by offline MALDI Mass Spectrometry. The identity of peaks 1-7 was furtherconfirmed by matching their migration time with that of standard,commercially available disaccharides. For example, the CE spectrum ofpeak 1 was collected from the CE analysis of total enzyme digest of UFHand a MALDI mass spectrum of peak 1 was generated. Mass spectra werecollected using parameters as outlined previously and calibratedexternally by using signals for protonated (RG)₁₉R and its complex witha nitrous acid-derived hexasaccharide of the sequence I_(2S)H_(NS,6S)I_(2S)H_(NS,6S) I_(2S)Man_(6S).

Results

As seen in FIG. 11A, eight peaks are seen in the CE spectrum. Each ofthe peaks labeled 1 through 8 has identical migration time in differentsamples. Following the collection of each peak and the purity check ofeach sample by re-injecting into CE, their mass was measured by off lineMALDI Mass Spectrometry. Peak 1 has the same mass as the trisulfatedheparin disaccharide ΔU_(2S),H_(NS,6S). The commercial ΔU_(2S),H_(NS,6S)from Sigma has the same migration time as peak 1 under identical CEconditions. This identifies peak 1 as ΔU_(2S),H_(NS,6S). In a similarmanner, the identities of the seven peaks from 2-7 in the compositionalanalysis digest of UFH were established. The results are shown in FIGS.3A and 3B-6A and 6B. The identity of each peak was further confirmed bymatching their migration time with that of standard, commerciallyavailable disaccharides. Peaks 2, 3, and 4 are disulfated disaccharides,and 5, 6, and 7 are monosulfated disaccharides.

A trace of the CE and MALDI mass spectrum of peak 2 was generated. Thispeak has the same mass as ΔU_(2S),H_(NS). Also the commercialΔU_(2S),H_(NS) from sigma has the same migration time as peak 2 underidentical CE conditions. This confirms that peak 2 is ΔU_(2S),H_(NS).

A trace of the CE and MALDI mass spectrum of peak 3 was generated. Ithas the same mass as ΔU,H_(NS,6S). Also the commercial ΔU,H_(NS,6S) fromsigma has the same migration time as peak 3 under identical CEconditions. This confirms that peak 3 is ΔU,H_(NS,6S).

A trace of the CE of peak 5 was generated. It has the same mass asΔU,H_(NS). Also the commercial ΔU,H_(NS) from sigma has the samemigration time as peak 5 under identical CE conditions. This confirmsthat peak 5 is ΔU,H_(NS).

A trace of the CE of peak 6 was generated. It has the same mass asΔU_(2S),H_(NAC). Also the commercial ΔU_(2S),H_(NAC) from sigma has thesame migration time as peak 6 under identical CE conditions. Thisconfirms that peak 6 is ΔU_(2S),H_(NAC).

A trace of the CE of peak 7 was generated. It has the same mass asΔU,H_(NAC,6S). Also the commercial ΔU,H_(NAC,6S) from sigma has the samemigration time as peak 7 under identical CE conditions. This confirmsthat peak 7 is ΔU,H_(NAC,6S).

In addition to the seven disaccharides, a tetrasaccharide (peak 8) inthe exhaustive digest of heparin with heparinase I, II, and III was alsoidentified. This tetrasaccharide was isolated, and its mass wasdetermined by MALDI Mass spectrometry. It had the same migration time inthe CE as the tetrasaccharide ΔUH_(NAC,6S)GH_(NS,3S,6S) that was part ofthe decasaccharideΔU_(2S)H_(NS,6S)I_(2S)H_(NS,6S)I_(2S)H_(NS,6S)IH_(NAC,6S)GH_(NS,3S,6S)whose structure was previously determined. This lead to the confirmationof peak 8 as the tetrasaccharide ΔUH_(NAC,6S)GH_(NS,3S,6S). This peak isresistant to further degradation with heparinases I, II, or III.

In addition to peaks 1-8, there was a small amount of unsulfateddisaccharides migrating much slower than the sulfated saccharides, asshown in FIG. 11B. The unsulfated disaccharides are estimated toconstitute <2% of UFH as shown in table 5 (below).

FIG. 12 shows the CE trace of the exhaustive digest of AT-10pentasaccharide ΔU_(2S)H_(NS,6S) ΔU_(2S)H_(NS,6S) ΔU_(2S)H_(NS,6S)IH_(NAC,6S) GH_(NS,3S,6S). Tetrasaccharide 8 in the exhaustive digest ofheparin has the same mass, and migration time asΔUH_(NAC,6S)GH_(NS,3S,6S). This confirms peak 8 asΔUH_(NAC,6S)GH_(NS,3S,6S).

Example 4 UV Response of Disaccharides 1-7

Introduction

The UV response factor (RF) of each of the disaccharides (1-7) wasdetermined to further identify the components of the disaccharides fromthe exhaustive digest. The RF for a disaccharide is defined as theamount of that disaccharide in ng that gives the same response as one ngof ΔU_(2S),H_(NS,6S).

Methods

Determining the Response Factor: RF for the different disaccharides wascalculated by measuring the UV response of 57 ng of each disaccharidesand normalizing it with that of ΔU_(2S),H_(NS,6S) as shown in table 5.

Results

Each of the seven disaccharides (1-7) has a-different extent of A₂₃₂ UVresponse.

There was an insufficient quantity of tetrasaccharide 8(ΔUH_(NAC,6S)GH_(NS,3S,6S)) to allow measurement of the A₂₃₂ UVabsorbance for this tetrasaccharide. Tetrasaccharides are expected tohave lower A₂₃₂ UV absorbance than disaccharides and henceΔUH_(NAC,6S)GH_(NS,3S,6S) has been assumed to have the same response asthe least responsive of the disaccharides, viz, 1 (ΔU_(2S),H_(NS,6S)).TABLE 5 Response for 57 ng Response Compound of disaccharides Factor(RF) 1 10421.1 1 (ΔU_(2S),H_(NS,6S)) 2 13551.45 0.769 (ΔU_(2S),H_(NS)) 314595.3 0.714 (ΔUH_(NS,6S)) 4 13551.45 0.769 (ΔU_(2S),H_(NAC,6S)) 523956.35 0.435 (ΔU,H_(NS)) 6 32363.55 0.322 (ΔU_(2S),H_(NAC)) 7 22903.350.455 (ΔU,H_(NAC,6S)) 8 10421.1 1 (ΔUH_(NAC,6S)GH_(NS,3S,6S))(assumption)

In Table 5 the response factor (RF) for peaks 1-8 was calculated. Thesecond column gives the UV absorbance at 232 nM for 57 nL injection of 1ng/nL concentration, i.e., 57 ng of the commercial standarddisaccharides (1-7). We could not measure the absorbance the UVabsorbance at 232 nM for known concentration of 8 for lack of sampleavailability in sufficient quantity. 8 is assumed to have the sameresponse as that of the least responsive of the 7 disaccharides, viz, 1.The third column gives the RF for each of the seven peaks, the RF for adisaccharide being defined as the fraction of a ng of it that gives thesame response as a ng of 1 (ΔU_(2S),H_(NS,6S)).

Example 5 Determination of Unsulfated Saccharides in Heparin

Introduction

Although heparin has been classified as a sulfated glycosaminoglycan, itcontains a minor amount of unsulfated saccharides. Procedures wereundertaken to determine the amount of unsulfated saccharides in heparin.Following this determination, the RF calculated for peaks 1-8 can beused to estimate the weight % of the sulfated building blocks of heparinfrom the AUC measured by CE.

Methods

A known weight of heparin subjected to exhaustive digestion with theenzyme cocktail was injected into the CE. The area under the curve (AUC)measured for peaks 1-8 in FIG. 11 is converted into the weight ofsulfated saccharides using the response for known amounts of peaks 1-8.The difference between the total amount of heparin saccharides injectedinto the CE, and the total amount of sulfated disaccharides andtetrasaccharide, gives the amount of unsulfated saccharides in thecomplete digestion of UFH. Multiplying the % relative AUC with the RFgives the corrected relative concentration or the % relative AUC ofpeaks 1-8 in terms of ΔU_(2S),H_(NS,6S).

Results

Table 6 shows the estimation of the unsulfated saccharides in heparin.As shown in table 6, unsulfated saccharides were determined toconstitute less than 2% of heparin. The unsulfated saccharides are nottaken into account in constructing the compositional analysis table ofheparin as explained below. Column 1 of table 7 gives the AUC measuredfor peaks 1-8. Column 2 gives the % relative AUC. Multiplying the %relative AUC with the RF gives the corrected relative concentration orthe % relative AUC of peaks 1-8 in terms of ΔU_(2S),H_(NS,6S). These arethen normalized to get the weight % of disaccharide peaks 1-7 andtetrasaccharide peak 8. As demonstrated here, construction of thiscompositional analysis table is independent of the concentration or theweight of the heparin digest analyzed by the CE. TABLE 6 Response Per 57ng Amount of saccharide Compound AUC sample in ng 1 7294.5 10421.1 39.9(ΔU_(2S),H_(NS,6S)) 2 1040.8 13551.45 4.4 (ΔU_(2S),H_(NS)) 3 1437.914595.3 5.3 (ΔUH_(NS,6S)) 4 379.3 13551.45 1.6 (ΔU_(2S),H_(NAC,6S)) 5685.1 23956.35 1.7 (ΔU,H_(NS)) 6 502.9 32363.55 0.9 (ΔU_(2S),H_(NAC)) 7482.3 22903.35 1.1 (ΔU,H_(NAC,6S)) 8 184.7 10421.1 1(ΔUH_(NAC,6S)GH_(NS,3S,6S)) (assumption) Total amount of the sulphatedsaccharides 55.9 from 1-8 Amount of unsulfated saccharides 57 − 55.9 =1.1 ng (1.9%)

Table 6 shows an estimation of the amount of unsulfated saccharides inthe exhaustive digestion of UFH with heparinases I, II, and III. Peaks1-7 are shown as known disaccharides as explained in FIGS. 2-6. Peak 8is tetrasaccharide ΔUH_(NAC,6S)GH_(NS,3S,6S) which is resistant tofurther degradation by either heparinases I, II, or III. The secondcolumn gives the area under the curve (AUC) measured for each of thepeaks for a 57 nL injection of 1 ng/nL concentration of compositionalanalysis digest of UFH. The third column gives the UV absorbance at 232nM for 57 nL injection of 1 ng/nL concentration of each of the 7commercial standard disaccharides (1-7). Column 4 gives the weight ofthe various sulfated saccharides in ng. The difference between their sum(55.9 ng) and the amount of heparin injected (57×1 =57 ng) gives theamount of unsulfated saccharides present in the compositional analysisdigest of UFH.

Example 6 Verification of Instrumentation and Completeness of DigestionIntroduction

To verify the instrumental reproducibility and to ascertain if thecompositional analysis digest is indeed complete under the enzymeconcentrations used, samples of UHF were digested in duplicate andanalyzed twice by CD. The results were then compared to determinewhether there was variability between runs.

Methods

UFH was digested as described in Example 1A, methods with either 1 μl or5 μl of enzyme cocktail (EC). Each sample was digested in duplicate, andeach digest was. analyzed twice by CE.

Results

Comparison of duplicate analysis of the same sample (UFH 1/1 with UFH1/2, UFH 2/1 with UFH 2/2, and UFH 3/2 with UFH 3/2) shows that there isgood instrumental reproducibility. Comparison of either UFH 1/1 or UFH1/2 with, UFH 2/1 or UFH 2/2 shows that there is minimal run-to-runvariation. Comparison of UFH digested with 1 μl of EC with UFH digestedwith 5 μl of EC illustrates that increasing the enzyme quantity does notchange the disaccharide profile appreciably. This confirms thatexhaustive digestion is reached by using 1 μl of EC as shown in FIG. 11.Compositional analysis of LMWH performed by CE as per the protocoloutlined in FIG. 11 and table 7 can be used to rigorously comparedifferent batches of LMWH. TABLE 7 % Relative Response Correctedrelative Compound AUC AUC Factor (RF) concentration Weight % 1 7294.560.7 1 60.7 70.9 (ΔU_(2S),H_(NS,6S)) 2 1040.8 8.7 0.769 6.7 7.8(ΔU_(2S),H_(NS)) 3 1437.9 12.0 0.714 8.6 10.0 (ΔUH_(NS,6S)) 4 379.3 3.20.769 2.5 2.9 (ΔU_(2S),H_(NAC,6S)) 5 685.1 5.7 0.435 2.5 2.9 (ΔU,H_(NS))6 502.9 4.2 0.322 1.4 1.6 (ΔU_(2S),H_(NAC)) 7 482.3 4.0 0.455 1.8 2.1(ΔU,H_(NAC,6S)) 8 184.7 1.5 1 1.5 1.8 (ΔUH_(NAC,6S)GH_(NS,3S,S))

Table 7 shows the values for Compositional Analysis for UFH. The areaunder the curve (AUV) was measured for each peak from the CE spectrum ofUFH digested with the enzyme cocktail as shown in FIG. 11. The responsefactor calculated for each saccharide as shown in table 6 was used tocalculate their corrected relative concentration in the enzyme digest.The last column gives the weight percentage of each of the buildingblock of UFH. The unsulfated saccharides, which constitute <2% of UFH,is not taken into consideration in constructing this compositionalanalysis table. As demonstrated here, construction of this compositionalanalysis table as shown by this method is independent of theconcentration or the weight of the heparin digest analyzed by the CE.

Example 7 Determining the Efficiency of AT-III Mediated Anti-Factor XaAntioagulant Action of Heparin: Correlation betweenIH_(NAC,6S)GH_(NS,3S,6S) and Anti-Xa Activity

Introduction

The quantification of ΔUH_(NAC,6S)GH_(NS,3S,6S) tetrasaccharide by CAMhas an additional role in estimating the efficacy of AT-III mediatedanti-factor Xa anticoagulant action of heparin.ΔUH_(NAC,6S)GH_(NS,3S,6S) is a part of the AT-III bindingpentasaccharide. Quantification of ΔUH_(NAC,6S)GH_(NS,3S,6S) is ameasure of the determination of the amount of AT-III bindingpentasaccharide present in heparin and thus it helps in the directmeasurement of anti-factor Xa mediated anticoagulation of heparin thatis dependent on the AT-III binding pentasaccharide domain of heparin.

Methods

UFH was size fractionated through P10 size exclusion column.Compositional analysis was performed on the resulting fractions toestimate their ΔUH_(NAC,6S)GH_(NS,3S,6S) content. These fractions werealso assayed for their anti-factor Xa activity.

Results

A plot of anti-factor Xa activity of different fractions as a functionof their ΔUH_(NAC,6S)GH_(NS,3S,6S) results in a straight line withr=0.91, as shown in FIG. 11. This indicates that the anti factor Xamediated anticoagulant action of heparins may be directly measured fromtheir ΔUH_(NAC,6S)GH_(NS,3S,6S) content. The data is also presented inTable 8. TABLE 8 Sample 1 2 3 4 5 6 7 8 ΔU_(2S),H_(NS,6S) ΔU_(2S),H_(NS)ΔU,H_(NS,6S) ΔU_(2S),H_(NAC,6S) ΔU,H_(NS) ΔU_(2S),H_(NAC) ΔUH_(NAC,6S)ΔUH_(NAC,6S)GH_(NS,3S,6S) UFH 70.9 7.8 10.0 2.9 2.9 1.6 2.1 1.8 1/1 1 μlEC UFH ½ 71.0 7.7 10.2 3.0 2.8 1.5 2.0 1.8 1 μl EC UFH 71.5 7.5 10.1 2.92.7 1.5 2.1 1.7 2/1 1 μl EC UFH 71.3 7.5 10.3 2.8 2.8 1.5 2.0 1.8 2/2 1μl EC UFH 72.0 7.3 10.0 2.8 2.8 1.6 1.8 1.7 3/1 5 μl EC UFH 72.2 7.5 9.92.7 2.7 1.6 1.7 1.7 3/2 5 μl EC

Table 8 shows a Compositional analysis of UFH performed by CE as per theprotocol outlined in FIG. 11 and table 7 can be used to rigorouslycompare different batches of LMWH. UFH was digested with either 1 μl or5 μl of enzyme cocktail (EC). Each sample was digested in duplicate andeach digest was analyzed in duplicate by CE. In all the samples,saccharide peaks 1-8 had the same migration time. Comparison ofduplicate analysis of the same sample (UFH 1/1 with UFH 1/2, UFH 2/1with UFH 2/2, and UFH 3/1 with UFH 3/2) shows that there is goodinstrumental reproducibility. Comparison of either UFH 1/1 or UFH 1/2with UFH 2/1 or UFH 2/2 shows that there is minimal run-to-runvariation. Comparison of UFH digested with 1 μpl of EC with UFH digestedwith 5 μl of EC illustrates that increasing the enzyme quantity does notchange the disaccharide profile appreciably showing that exhaustivedigestion is reached by using 1 μl of EC as shown in FIG. 11.

Example 8 Generation of LMWH Fractions and Characterization ofBiological Activity

Methods:

LMWH fractions MS 57-1 through MS 57-4 and MS 59-1 to MS59-4 wereprepared by treating UFH with 200 μg of Heparinase III (as describedabove) and passing the resulting product through a P10 column.

LMWH fractions MS56-1 through MS56-4 were prepared by treating UFH with1000 μg of Heparinase III, and passing the resulting product through aP10 column.

LMWH fractions MS60-1 through MS 60-3, and MS55-1 through MS55-4 weregenerated by treating Fraction 2 with 200 μg of Heparinase III, andpassing the resulting product through a P10 column.

LMWH fractions MS 66-1 and MS66-2 were prepared by treating Fraction 2with 1000 μg of Heparinase III, and passing the resulting productthrough a P10 column.

Fraction 1 is the high molecular weight heparin that is precipitatedupon treating UFH with Barium acetate at room temperature, as describedin the Volpi reference described above. TABLE 9 Generation of Heparin ofSelected MW/Charge/Biological Properties Wt. Average anti-Xa anti-IIaXa/ Method Sample (mg) MW PD IU/mg IU/mg IIa Wt % 1 Wt % 8 Wt % 2 Wt % 3Wt % 4 Wt % 5 Wt % 6 Wt % 7 Fraction 300 <8000 400.0 75 5.3 70.0 4.5 7.811.1 3.2 2.4 1.4 0 2 Fraction 150 8000-14,000 106.6 54.5 2.0 87.2 0 9.01.6 0.4 1.8 0 0 1 UFH, MS57-1 130 8743 ± 177 1.1 292.9 72.3 4.0 77.8 3.53.0 10.7 1.4 0.3 0.2 3.0 200 μg MS57-2 140 6010 ± 138 1.1 186 33.33 5.675.5 3.5 4.0 10.7 1.6 0.5 0.4 3.8 HepIII, MS57-3 60 3000-5000   70.8 073.9 2.8 4.9 10.2 1.8 1.2 0.6 5.0 P10 MS57-4 10 <3000 42.6 0 73.9 1.54.4 10.0 1.3 2.0 0.9 0.4 MS59-1 130  8800 255 29.2 8.7 73.1 4.3 5.8 11.52.5 0 0.5 2.4 MS59-2 130  6500 255 54.5 4.7 77.8 3.8 3.2 10.3 1.3 0.40.3 3.4 MS59-3 70 3000-5000   54 0 75.3 2.3 4.6 10.2 1.9 0.8 0.8 3.8MS59-4 30 <3000 15 0 71.3 1.9 5.7 9.4 1.9 1.8 2.0 6.2 UFH, MS56-1 609204 ± 192 1.2 261.4 50 5.2 78.8 4.7 3.3 9.3 1.7 0.2 0.3 2.2 1000 μgMS56-2 40 6000 ± 146 1.2 123.8 75 1.7 70.7 3.8 7.0 10.5 2.8 1.2 1.0 2.9HepIII- MS56-3 100 3000-5000   126.9 36.4 3.5 76.4 3.0 4.1 11.0 1.5 0.50.3 3.6 P10 MS56-4 80 <3000 31 0 78.7 0 5.0 12.0 2.1 0 0 0 FractionMS60-1 110 7224 ± 139 1.2 347.4 58.33 6.0 68.8 5.0 6.0 13.1 2.8 0.4 0.53.0 2, 200 MS60-2 140 5736 ± 238 1.1 353.6 75 1.5 69.9 3.8 6.2 13.0 2.10.8 0.7 3.8 μg MS60-3 140 <5000 51 0 65.9 0 5.7 12.7 2.2 3.7 1.7 8.4HepIII, MS55-1 140 6505 ± 100 1.1 335 60.0 5.6 68.6 6.5 5.5 13.9 3.3 0.30.3 2.1 P10 MS55-2 130.0 5964 ± 87  1.1 201.2 54.6 3.7 74.4 4.9 3.7 11.32.6 0 0 3.5 Fraction MS55-3 50.0 3000-5000   11.6 0 76.1 2.3 4.4 9.8 1.70.8 0.6 4.2 2, 1000 MS55-4 30 <3000 15.6 0 73.4 0 5.0 9.8 2.0 1.8 1.06.5 μg MS66-1 150 6470 ± 142 1.2 250.1 100 2.5 75.0 3.6 7.5 8.6 8.6 3.81.0 0.3 HepIII, MS66-2 150 5592 ± 159 1.1 206.0 20.8 6.0 74.0 3.6 7.89.2 3.4 1.1 1.0 0.2 P10

Fraction 2 is the second fraction of a lower MW that is precipitatedupon keeping the Barium acetate treated Heparin at 4C. This is thefraction used according to the methods of the invention.

The subcutaneous and in vivo absorption profiles of MS55-2 weremeasured. The absorption profile of MS55-2 was compared with that ofcommercially available LMWH Ardeparin, and Enoxaparin. The anti-Xaactivity of the various heparin species were also assayed for their invitro biological activity.

Results:

Table 9 provides the compositional and functional analysis of the LMWHpreparations prepared according to the invention and of the controlfraction 1. The table lists the MW, in vitro activity, and compositionof the various fractions.

MS55-2 showed very similar pharmacokinetics to that of enoxaparin asevident in the comparable absorption and elimination phase as well asT_(max). The bioavailability and Peak concentration were comparableamong all three LMWHs tested by Subcutaneous injection. Whenadministered by the IV route, the initial anti-Xa activity is muchhigher for MS55-2 in comparison with Aredeparin. Again, thebioavailability between the two LMWHs was almost identical. Bothardeparin and MS55-2 exhibited exponential decrease in anti-Xa activity,and thus the elimination follows first-order pharmacokinetics.

The anti-Xa activity of MS55-2 was observed to be 205 IU/mg. This ishigher than the LMWHs such as Enoxaparin (135 lU/mg), Ardeparin (93IU/mg) that are currently available in the United States. The resultsare also shown in FIG. 13.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by examples provided, since theexamples are intended as a single illustration of one aspect of theinvention and other functionally equivalent embodiments are within thescope of the invention. Various modifications of the invention inaddition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description and fall withinthe scope of the appended claims. The advantages and objects of theinvention are not necessarily encompassed by each embodiment of theinvention.

All references, patents and patent publications that are recited in thisapplication are incorporated in their entirety herein by reference.

1. A method for analyzing or processing a heparin sample comprising amixture of structurally diverse polysaccharides, comprising the stepsof: applying a separation method to the mixture; determining the amountof a signature component in the sample; and making a determination aboutthe sample based upon a comparison of the amount of the signaturecomponent in the sample to a reference database for a heparin, tothereby analyze the sample.
 2. The method of claim 1, further comprisingselecting the sample based upon the determination.
 3. The method ofclaim 1, wherein the heparin sample is a pharmaceutical product.
 4. Themethod of claim 1, wherein the heparin sample is a pharmaceutical gradeproduct governed by the USP.
 5. The method of claim 1, wherein theheparin sample is a commercial product.
 6. The method of claim 1,wherein the heparin sample is a low molecular weight heparin (LMWH)sample.
 7. The method of claim 6, wherein the LMWH sample is made by aprocess comprising Θ-eliminative cleavage with benzyl ester of heparinby alkaline treatment.
 8. The method of claim 6, wherein the LMWH sampleis an enoxaparin sample.
 9. The method of claim 6, wherein the LMWHsample is made by a process comprising partial nitrous depolymerizationof unfractionated heparin.
 10. The method of claim 6, wherein the LMWHsample is a fragmin sample.
 11. The method of claim 6, wherein the LMWHsample is made by a process comprising salt precipitation of anunfractionated heparin in a solvent that produces a first high molecularweight fraction and a second fraction of LMWH, and processing the secondfraction to produce a concentrated LMWH preparation.
 12. The method ofclaim 11, wherein said process further comprises digestion with aheparin degrading enzyme.
 13. The method of claim 12, wherein theheparin degrading enzyme is heparinase III.
 14. The method of claim 13,wherein at least one histidine residue selected from the groupconsisting of His 36, His105, His110, His139, His152, His225, His234,His241, His424, His469, and His539 of the heparinase III has beensubstituted.
 15. The method of claim 14, wherein the at least onehistidine residue has been substituted with alanine, serine, tyrosine,threonine, or lysine.
 16. The method of claim 15, wherein the at leastone histidine residue is His225 that has been substituted with alanine.17. The method of claim 1, wherein the reference database is embodied ina computer readable medium. 18-81. (canceled)
 82. A method of making adetermination about the quality of a sample, comprising: analyzinginformation obtained by a method of claim 1 to determine if the samplehas a signature component in the preselected range. 83-90. (canceled)91. A method of analyzing or processing a heparin sample comprising amixture of structurally diverse polysaccharides, comprising the stepsof: contacting the heparin sample with one or more heparin degradingenzyme, determining the amount of a signature component in the sampleusing a separation method, and comparing the amount of the signaturecomponent to a reference database for heparin, to thereby analyze orprocess the heparin sample.
 92. (canceled)
 93. A method for analyzing orprocessing a heparin sample comprising a mixture of structurally diversepolysaccharides, comprising the steps of: applying a separation methodto the mixture; determining the amount of a signature component in thesample, wherein the amount of signature component is calculated by theequation: PRA=RF×AUC_(%R), wherein PRA=percent relative amount of asignature component, RF=response factor, AUC_(%R)=percent relative AUC[100×AUC_(C))/AUC_(T))], AUC_(C)=area under the curve for one signaturecomponent, AUC_(T)=sum of area under the curve for all signaturecomponents, and making a determination about the sample based upon acomparison of the amount of the signature component in the sample to areference database for a heparin, to thereby analyze the sample. 94.(canceled)